Pulse Duplicator System User Manual

Transcription

1 Pulse Duplicator System User Manual Pulse Duplicator System Including Data Acquisition System Document No V1.3 ViVitro Labs Inc Page 1 of 118

2 ViVitro Labs Pulse Duplicator System Designed and Distributed by ViVitro Labs Inc., BC, Canada Disclaimer and Limitation of Responsibility ViVitro Labs Inc. assumes no responsibility for any damage, loss, or claims which may result from a failure to follow the instructions contained in this manual. Additionally, ViVitro Labs Inc. assumes no responsibility as a result of direct or derived injury, data loss, system malfunction which occurs at the fault of misuse, alternation of the system or incorrect application of use so advised or directed by ViVitro Labs. Copyright Copyright 2014 by ViVitro Labs Inc. All rights reserved. Published by ViVitro Labs Inc. Victoria, British Columbia, Canada Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this document, they make no representation or warranties with respect to the accuracy or completeness of the contents of this document and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales material. The advice and strategies contained herein may not be suitable for your situation. Neither the Publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential or other damages. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise without the prior written permission of the Publisher. Requests to the Publisher for permission should be addressed to: ViVitro Labs Inc. 455 Boleskine Rd Victoria, BC, Canada, V8Z 1E7 info@vivitrolabs.com tel toll free fax Document No V1.3 ViVitro Labs Inc Page 2 of 60

3 Table of Contents 1. About ViVitro Labs Cautions and Warnings About this Manual About the Pulse Duplicator System System Hardware Hardware Features ViViTest Software Software Features Hardware System Software Requirements Upgrade Installation Instructions About the AR Series SuperPump System Hardware Features SuperPump Initial Set-up Procedures Unpacking List of Components Installing the SuperPump Front Panel Rear Panel Electrical Requirements Fuses Running the SuperPump System Waveform Source Initialize System Select Waveform Set Sync Delay Change the Cycle Count Pump Operation SuperPump Faults and Alarms Specifications Pump Mounting Details Feet Bolt Pattern Pump Cylinder Mounting Face Bolt Pattern...22 Document No V1.3 ViVitro Labs Inc Page 3 of 118

4 5. Valve Mounting and Molding Procedure Rubber Ring Moulding Considerations GI-1100 Silicone Rubber Ring Moulding Procedure Preparation of RTV silicon rubber Mixing De-aerating mixture Pulse Duplicator System Assembly Unpacking List of Components SuperPump Attaching the VIA and the Ventricle Chamber Attaching the Ventricle Chamber Filling the Ventricle Chamber, VIA (if installed) and Pump Head Accessories Table Installation Atrium Chamber Installation Aortic Assembly Installation Flow Probe(s) Initial Operational Check Installation of Flow Probes Installing the Aortic Flow Probe Installing the Mitral Flow Probe Aortic Compliance Chamber Installation Aortic Room Compliance Chamber Peripheral Resistance Controller Heat Exchanger System Installation (Optional) Silicon Hose Installation Electrical Connections Electrical Connection Diagram Testing Set-up Procedures Initial Assembly Procedure Installation of Valves Priming / Preparing the Chambers Air Bubble Removal Characteristic Compliance and Liquid Level Adjustment Peripheral Resistance Adjustment...45 Document No V1.3 ViVitro Labs Inc Page 4 of 118

5 7.7 Pressure Waveform Adjustment Viscoelastic Imedance Adapter (VIA) adjustments Mean Atrial Pressure Transducer Settings System Preparation for Calibration Pump Calibration Operational Check Drive Waveform Generator Operational Check Calibration of Flow Probe Methods Flow Meter Operational Check Null, Balance and Zero System Check Calibration Procedure for Aortic Flow Probe Calibration Preparation of Mitral Flow Probe Calibration of Pressure Measurement System (AP9991) Initial Setup ViViGen Software Installation Method Icons Status Request New Waveform Generation ViViTest Software Operation Calibration Process Overview Calibration Wizard Navigation Pump Calibration Flow Meter Calibrating the Flow Meter Pressure Calibration Calibrating and Zeroing the Sensors Zero Calibration Measuring Sensor Zero References Finishing Calibration Updating the Calibration File Acquisition Process Standard Valve Verification Re-zero Sensors Analyze Process Crossovers and Interval Identification...70 Document No V1.3 ViVitro Labs Inc Page 5 of 118

6 10.13 Producing Data Output Derived Flow Measurements Useful Icons Compare Process Comparing Groups of Trials Auto-scaling and Zooming Handling Compare Results Review Process I/O Module Re-initialization Running a Report Taking a Snapshot Exporting CSV file data ANA file Log File Waveform Design Software Interface Orientation Top Level Toolbar Process Selection and Secondary Buttons Support Information Area Main Process Controls Data Dashboard Process Display Process Output Help System Maintenance Required Fluid Draining Fluid Cleaning Acrylic Fluid Aorta Replacement Ventricle Member Replacement Piston Seal Replacement Appendix A Supplemental Component Notes Appendix B Installing LVOT Accessories (Optional)...93 Document No V1.3 ViVitro Labs Inc Page 6 of 118

7 15. Appendix C LVOT Simulation Assembly (Optional) Appendix D Components and Parts Lists SuperPump Model Left Heart Data Acquisition System Flow Measuring Optional System Components Appendix E Glossary Appendix F Pump Calibration Procedure Resetting Values Determing Interal Pump Calibration Value External Gain Value Determining External Pump Calibration Value Appendix G Calculations Appendix H Step By Step Guide Definitions Summary Test (ref ISO Annex N) Cautions Pulsatile Flow Testing System Setup Calibration Inserting Valves Filling Pulse Duplicator Zeroing the System Tuning System Data Capture Moving Markers Recoding Data Drain / Remove Valve Data Quality Assurance Reporting Test Results Maintenance Appendix I End User License Agreement Document No V1.3 ViVitro Labs Inc Page 7 of 118

8 21.1 Grant of License Ownership of the Software / Restrictions on Copying Other Restrictions on Use Term Responsibilty for Selection and Use of Software Limited Warranty, Exceptions & Disclaimers European Software Directive General Provisions Canadian Sales Appendix J Wetted Materials Document No V1.3 ViVitro Labs Inc Page 8 of 118

9 1. About ViVitro Labs ViVitro Labs Inc. offers industry leading cardiovascular test equipment and related laboratory testing services. Hundreds of organizations in over 39 countries for 30+ years have trusted ViVitro s expertise, accuracy, and quality for their cardiovascular device testing. As the developer of the world s first Pulse Duplicator, the ViVitro name has been synonymous with cardiovascular device testing equipment. ViVitro hardware and software products have been used by leading R&D facilities and academic labs worldwide, and its equipment and testing methods being cited in hundreds of peer reviewed publications The ViVitro Labs Pulse Duplicator System (in particular the ViVitro Labs SuperPump) has become a worldwide standard to simulate physiological heart conditions in academic research applications. ViVitro products are manufactured by StarFish Medical in an ISO 13485:2003 certified manufacturing facility. ViVitro Labs Inc., also holds ISO/IEC accreditation for laboratory testing services endorsed by A2LA and based on ISO This scope of accreditation includes the physical and mechanical testing of heart valve substitutes including durability, hydrodynamic, and flow visualization testing. ViVitro Labs is widely recognized as the authority on cardiovascular device testing. ViVitro Laboratory Services are engaged worldwide for hydrodynamic, durability, and flow visualization testing of heart valves and other cardiac devices. ViVitro has been the trusted name in regulatory approvals for over 30 years and is renowned for its proven success from product development testing through to full regulatory submission. ViVitro Labs is a member of the ISO 5840 standards committee and is actively engaged in developing regulatory requirements. Leveraging this intimate knowledge of the standard, ViVitro Labs ensures that test protocols will meet ever changing regulatory requirements. ViVitro s Laboratory Testing Services offers an ISO/IEC certified lab using ViVitro equipment, to conduct 3 rd party independent testing. ViVitro s accredited testing lab is governed by a mature Quality Management System (QMS) certified to meet the ISO This gives assurance to stakeholders and regulatory bodies that results are obtained by qualified personnel using traceable calibrated equipment and up to-date test methods, all supervised by a quality assurance department. Document No V1.3 ViVitro Labs Inc Page 9 of 118

10 1.1 Cautions and Warnings After you receive your Pulse Duplicator, ensure that you let the electrical enclosures sit indoors at room temperature for at least one day before plugging them in. The systems must not be used in environments at higher than 75% humidity. Read all of the instructions before operating the SuperPump This instrument is intended for research use only and must be operated by trained lab personnel. To ensure adequate cooling of the SuperPump controller and pump, place on a clean, flat surface, and be sure that there is at least 10 cm (4 in) of clearance around each side of the controller and motor end of the pump. Make sure that the fan vent is not blocked. Always connect the power supply to a 3-prong, grounded AC outlet rated 4A, V using the AC power cord provided with the SuperPump. Do not use an adapter to an ungrounded outlet. Before plugging the instrument in, be sure that the correct fuses are installed. See section 3.9. Electrical devices pose the risk of electric shock. To reduce the risk of shock, do not open any covers that are fastened with screws. While SuperPump is designed to withstand spills on its exterior surface, do not allow fluids to enter the interior of the instrument. In the event of such a spill, disconnect the power cable before cleaning up. Do not operate SuperPump in extreme humidity or in conditions that can create condensation. Protect the instrument against dust and moisture, and avoid physical shock and strong forces. Clean the controller and pump exterior with a dry cloth. Disconnect power before cleaning Use caution when moving the controller and/or pump. Disconnect power and pump cable before moving the system to a new location. Do not use the SuperPump system if there is evidence of physical damage or if the system has been dropped or subject to impact. Do not attempt to service the product beyond what has been described in this manual. All other servicing should be referred to qualified service personnel. 1.2 About this Manual This document describes the Pulse Duplicator System s procedures for: 1. Assembly of the system s hardware. 2. Connection of the system s electrical components. 3. Calibration and system setup. 4. Operator procedures for the system s daily use. 5. Operator procedures for ViViTest Software. 6. Operator procedures for ViViGen Software. 7. Optional valve and other cardiac structure mounting procedures for use within the Model Left Heart. NOTE: The user may opt to manipulate the system to achieve alternate states/results/configurations. Should the user carry this out it should be noted that the pressures and flows are kept within the limitations of the system to ensure the system is not overloaded. Document No V1.3 ViVitro Labs Inc Page 10 of 118

11 2. About the Pulse Duplicator System 2.1 System Hardware Pulse Duplicator System User Manual The ViVitro Labs Inc. Pulse Duplicator System assesses heart valve function under simulated cardiac conditions. The system simulates the function of the heart s ventricle which generates pulsatile flow through a prosthetic heart valve in a ViVitro Labs Model Left Heart. A Pulse Duplicator simulates physiological or other complex pressure variations which (when connected to the ViViTest software via sensors) provides detailed data return on prosthetic heart valve(s) and cardiac system performance. The ViVitro Labs Pulse Duplicator System is ideal for testing a variety of prosthetic heart valves. Standard and optional hardware components facilitate the mounting of mechanical, stented and stentless bioprostheses, percutaneous devices. The system is also suitable for other related cardiovascular purposes such as measurements of root compliance (in conjunction with video recording) or average coronary flow. 2.2 Hardware Features The Pulse Duplicator System from ViVitro Labs Inc. features include: Assessment of heart valve performance and function under simulated cardiac conditions. View and monitor heart valves under simulated conditions via: o transparent parts and view ports to observe and video record valve function and adjacent flow fields; o aids in flow visualization studies; o thin acrylic windows permit ultrasound transmission for interrogation of flow fields in aortic and mitral regions; o transducer sites to measure wall pressures in the atrium, ventricle, aortic outflow tract, downstream of the aortic valve and upstream of the aortic and mitral valves. Control pulsatile fluid flows to simulate various cardiac flow conditions to include arrhythmia, normal, hypo and hypertensive states at various cardiac outputs and beat rates. Peripheral resistance and supplementary characteristic compliances to simulate cardiovascular character and resistance. Control, create, download and playback cardiac waveforms at various physiological states and lower frequencies. Quality data acquisition which meets verification and testing in accordance with ISO requirements and aids in the collection of data to meet ISO 5840 to FDA requirements through the use of ViViTest software. Optional components enable: o Testing of various cardiac valve types and cardiac structures. o Viscoelastic simulated behavior to produce realistic ventricle pressure waveforms. o Operate test fluid at 37ºC to simulate physiological conditions. Document No V1.3 ViVitro Labs Inc Page 11 of 118

12 2.3 ViViTest Software This document describes the ViViTest software program designed to acquire data from the ViVitro Labs Pulse Duplicator System in characterizing heart valves. This document along with hardware operations manuals for the various elements of the Pulse Duplicator System should be considered as the primary reference document for operations of the ViViTest software package. ViViTest is designed for monitoring, acquisition and analysis of data generated by laboratory systems for assessment of heart valves. This software is primarily developed for the Pulse Duplicator System from ViVitro Labs. For use with other systems please contact ViVitro Labs for technical assistance. 2.4 Software Features The software consists of four primary processes: Waveform Acquisition Waveform Analysis Waveform Comparison Waveform Review and one secondary utility: Sensor Calibration The ViViTest software is shipped pre-installed on a PC. Components: Laptop or Desktop PC (with monitor, keyboard and mouse) I/O Module.NET 3.5 framework 2.5 Hardware System The ViViTest software is shipped pre-installed on a PC. Components: Laptop or Desktop PC (with monitor, keyboard and mouse) I/O Module.NET 3.5 framework The ViViTest software application is designed to run on the following minimum hardware specifications: LCD monitor capable of 1600 x 900 resolution High throughput video card I/O Module 2GB RAM 38GB Hard drive Pentium GHz Processor (single core) or better Document No V1.3 ViVitro Labs Inc Page 12 of 118

13 2.6 Software Requirements For the software to run smoothly the following software is required to be installed on the computer: Microsoft Windows XP Professional SP3, Windows 7, Windows 8.NET 3.5 framework. 2.7 Upgrade Installation Instructions As the software upgrades are released you will be notified via . Instructions: 1. Download updater installer file. 2. Select the single comprehensive installer executable. 3. An installation Wizard will guide you through the installation steps. 4. Unless provided the installation key should be left blank. NOTE: With some upgrade releases there may be the necessity to upgrade the I/O Module files and/or the.net files. The Wizard will guide you through this. Should you incur problems, contact ViVitro Labs directly. Document No V1.3 ViVitro Labs Inc Page 13 of 118

14 3. About the AR Series SuperPump 3.1 System Hardware Pulse Duplicator System User Manual The Advanced Research series SuperPump System made by ViVitro Labs Inc. consists of a digital amplifier with stroke volume display and preprogrammed waveforms driving a piston-incylinder pump. The pump piston is located by a ball screw/servo motor/encoder unit driven by a motor controller which compares actual position against the desired position defined by the input waveform. The precision of the ball screw and the resolution of the encoder ensure accurate positioning of the piston resulting in the desired pulsatile flow. The controller can be used stand alone by selecting one of the built in waveforms or an external signal can be input. ViVigen software is included with the pump to allow the generation of custom waveforms which can then be uploaded to the controller via the built in USB port. The SuperPump is designed for use as part of the ViVitro Pulse Duplicator and includes the required inputs and outputs for this. These same I/O including analog outputs for position, dl/dt (flow) and the sync pulse make the SuperPump adaptable to any custom test apparatus. The ViVitro Labs AR series SuperPump is ideal for testing a variety of cardiovascular devices. 3.2 Features The SuperPump-AR series from ViVitro Labs Inc. features include: Digital precision control of a pump-in-cylinder designed linear actuator used to generate physiological flows; Stand-alone capability with five (5) preprogrammed cardiac waveforms created by user; Range of capable rates are 4 to 200 beats per minute; Range of stroke volume 0 to 150 ml; Digital controlled and created waveforms by ViViGen or ViViTest software; Control, create, input or store cardiac waveforms at various physiological states and frequencies through ViVitro software or other std waveform generating equipment; Manufactured by an ISO accredited facility designed to meet regulatory requirements (ISO 5840 and FDA) or research needs. NOTE: The user may opt to manipulate the system to achieve alternate states/results/configurations. Should the user carry this out it should be noted that the pressures and flows are kept within the limitations of the system to ensure the system is not overloaded. 3.3 SuperPump Initial Set-up Procedures The SuperPump System has several steps to the main mechanical sub-assemblies, and a few steps for the electrical components installation/configuration. NOTE: It is critical for the assembly steps to be followed in order to assure successful operation. Document No V1.3 ViVitro Labs Inc Page 14 of 118

15 3.4 Unpacking List of Components The SuperPump AR arrives in 2 containers; a wooden shipping crate for the pump and a cardboard container for the controller. Inspect the shipping containers for external damage. Retain the shipping carton and packing materials for possible re-shipment. Inspect the system components for damage. If there is evidence of damage contact the carrier, initiate a damage claim, and inform ViVitro Labs. The SuperPump arrives pre-assembled with the following parts: Mounting base Linear Actuator with Cable Standard Pump Head Actuator Extension Cable SuperPump Controller USB flash drive with ViVigen software 3.5 Installing the SuperPump The SuperPump is supplied mounted to a rigid plastic base for use on a bench top. Optionally the pump may be mounted directly to a structure or test apparatus using #10-32 fasteners through the mounting feet of the pump. See bolt pattern layout in Section 4.9. It is recommended that the SuperPump be mounted horizontally. The back side of the pump cylinder is provided with a drain, however vertical mounting of the pump can result in water entering the ball screw/motor causing an electrical hazard. The controller should be located away from the pump to avoid any water or test fluid. An extension cable is provided to allow extra separation if required. It is recommended that the pump act on the test fluid through a membrane to maintain isolation between working and test fluid. The piston should not be directly exposed to blood analog solution or saline. Distilled water is recommended as the working fluid. 3.6 Front Panel The front panel consists of 2 controls and the display. The controls are rotary encoders with push switches. To the left of the display is the menu knob which allows scrolling by turning the knob and selection by pressing the knob. To the right of the display is the amplitude knob which adjusts the amplitude of the output waveform and starts and stops the pump by pressing the knob Document No V1.3 ViVitro Labs Inc Page 15 of 118

16 3.7 Rear Panel The rear panel contains all of the connectors. From left to right: Waveform In: BNC connector used when an external signal is used as the waveform Position: BNC connector analog output signal for the piston position dl/dt: BNC connector analog output signal proportional to instantaneous flow rate Sync Out: BNC connector 5V TTL signal triggered at the rising zero crossing of the waveform. The timing of the signal can be adjusted to any point in the waveform Troubleshooting port : no user accessible functions USB: For uploading custom waveforms created using ViVigen software or for use with ViVitest when the SuperPump is used as part of the Pulse Duplicator Motor: Cable connection to the pump Cooling fan Power input: Power cord, fuse holder and main power on/off 3.8 Electrical Requirements ViVitro Labs Inc. will endeavor to have your system s components adapted to meet your country s power supply, however, as an extra precaution, it is advised that all equipment designated to be connected to a power supply be checked prior to use. Use only the power cord supplied with your SuperPump. The controller must be plugged into a grounded outlet rated for 300W. 3.9 Fuses Two fuses are supplied and required for the SuperPump Controller. They are located in the fuse drawer on the power input module on the back of the controller. At 230VAC use two 2A, 5mm x 20mm 230V fuses. At 120VAC use two 4A, 5mm x 20mm 230V fuses. Contact ViVitro Labs if you require replacement fuses. Document No V1.3 ViVitro Labs Inc Page 16 of 118

17 4. Running the SuperPump System 4.1 Waveform Source Pulse Duplicator System User Manual The SuperPump may be operated using either: a) USB connection to a computer running ViViTest Software see ViViTest user manual b) Internally stored waveform see section 8 ViVigen c) Externally generated waveform If an external waveform is to be used, plug an appropriate BNC cable from the waveform source to the Waveform In port on the back of the SuperPump Controller (sec 2.4). The external waveform should conform to the following specifications: Amplitude: 10.0 Vpp (±5.0 Vpp) maximum. Any waveform that exceeds this amplitude will not be accurately represented in the pump movement. Rate [Frequency]: 4 bpm (beats per minute) minimum to 200 bpm maximum. [0.067 Hz to 3.33 Hz] NOTE: Do not input waveforms with frequency greater than the listed maximum. Form: Bi-polar. The waveform should be centered about 0 V (ground), without D.C. offset, unless offset is intended for recreating a particular wave shape. The peak-topeak limits with offset must still remain within ±5.0 Vpp relative to ground. Noise and Distortion: both noise and distortion will affect the accuracy of the pump movement; therefore the signal source should be clean, with THD+N (total harmonic distortion and noise) below 5%. Note: the maximum velocity of the piston is limited to approximately 60mm/sec. As amplitude and frequency are increased the velocity will also increase. If the pump cannot match the desired waveform a pump error will result. In this case either the maximum slope of the waveform must be reduced or the frequency or amplitude must be reduced. Document No V1.3 ViVitro Labs Inc Page 17 of 118

18 4.2 Initialize System Turn on the power switch on the back of the Controller. The screen on the front of the Controller will light up and indicate that it is Initializing. It will then automatically begin a Homing Pump sequence. Firmware Version Firmware Programming Date Menu Selector Amplitude Dial After automatically Homing Pump, the screen should appear as below. The most recently used waveform source will appear in the Current Waveform section. Current Amplitude Counter Current Stroke Volume Current Waveform Sync Output Delay Document No V1.3 ViVitro Labs Inc Page 18 of 118

19 4.3 Select Waveform Press the Menu Selector Dial. Turn the Menu dial until 1 Waveform Source appears as below % Gain 0.0 ml/stroke 1 Waveform Source While the message Waveform Source is on the screen, press the dial again. The screen will now indicate the most recently used waveform source % Gain 0.0 ml/stroke Wfm: External If the current waveform source is desired press the dial again to escape the waveform selector. If a stored waveform is desired turn the dial clockwise and press the dial when you have chosen the correct waveform. Refer to Section 9 to load standard waveforms, edit, or create waveforms and load them onto the SuperPump Controller % Gain 0.0 ml/stroke Wfm: FDA 4.4 Set Sync Delay Press the Menu Selector Dial. Turn the menu knob until 2 Sync Pulse Delay is on the screen, press the dial again. The screen will now indicate the current sync delay in msec % Gain 0.0 ml/stroke 2 Sync Pulse Delay To adjust the delay turn the dial until the correct delay is displayed and then press the dial. 4.5 Change the Cycle Count Press the Menu Selector Dial. Turn the menu knob until 3 Totalizer is on the screen, press the dial again % Gain 0.0 ml/stroke 3 Totalizer To adjust the counter turn the dial until the correct value is displayed and then press the dial % Gain 0.0 ml/stroke 3 Totalizer Document No V1.3 ViVitro Labs Inc Page 19 of 118

20 4.6 Pump Operation % Gain 0.0 ml/stroke FDA 0 msec Turn the Amplitude Dial until the desired stroke volume is reached. For example: % Gain 73.4 ml/stroke FDA 0 msec To stop the pump either turn the Amplitude Dial to 0 or press the Amplitude Dial. In this case an Emergency Stop alarm will appear. To clear this alarm, press the Amplitude Dial. The pump will automatically perform a Homing Sequence. 4.7 SuperPump Faults and Alarms Emergency Stop If case of an Emergency Stop, press the Amplitude Dial to clear the alarm and Home the pump % Gain 0.0 ml/stroke Emergency Stop! Limit Switch The SuperPump actuator contains a factory-set limit sensor for the maximum push and pull stroke. In case of a Limit reached alarm, press the Amplitude Dial to clear the alarm and Home the pump % Gain ml/stroke Limit reached! Pump Error If the Controller loses communication with the Pump or if the Pump is unable to move, a Pump error message will appear. If this occurs, 1. turn off the system 2. check that the cables are properly installed 3. ensure that the pump is free of mechanical interference and damage 4. turn the controller on and allow it to initialize normally 5. If the alarm persists, contact the manufacturer % Gain 0.0 ml/stroke Pump Error! Document No V1.3 ViVitro Labs Inc Page 20 of 118

21 4.8 Specifications Pump Pump bore diameter Nominal 69.85mm (2.750 ) Pump stroke Displacement Rate Inputs External Waveform Outputs Position DL/dt Sync Power Requirements Voltage Power Dimensions Controller Pump 0-40mm (±20mm) 0-150mL 4 to 200 beats per minute 0-10 Vpp Hz to 3.33 Hz (4 to 200 beats per minute) -5 to 5 V (200mV/mm) -5 to 5 V (539 ml/s/v) +5V TTL LE at rising zero crossing VAC / 50-60Hz Max 300W 11 x 48 x 33 cm 15 x 55 x 17 cm Document No V1.3 ViVitro Labs Inc Page 21 of 118

22 4.9 Pump Mounting Details Feet Bolt Pattern 4.10 Pump Cylinder Mounting Face Bolt Pattern Document No V1.3 ViVitro Labs Inc Page 22 of 118

23 5. Valve Mounting and Molding Procedure 5.1 Rubber Ring Moulding Considerations Pulse Duplicator System User Manual Silicone rubber rings are recommended for mounting heart valves or other test specimens. The rubber rings provide a fluid seal between the device under test and the test system flanges. Since each size and type of test device differs in geometry, a unique rubber ring is required in order to match and seal the appropriate boundaries on the device. The rubber ring should not interfere with the action of the device being tested. The considerations discussed here apply to the design of rubber rings for mounting mechanical or tissue valves, with or without sewing rings. Molding jig components for valves are shown coloured in the following illustration. The jig components can be separated and adjusted by loosening bolt A and thumb screws C & D. The custom pedestal (Light GREY) is attached to the base (PURPLE) with a bolt (A). The pedestal can be the actual valve or a replica of the outer dimensions to which the rubber ring seals. It can be made of Delrin which has good mold release ability. When a valve or valve Document No V1.3 ViVitro Labs Inc Page 23 of 118

24 component is used as a mold, a thin layer of Vaseline coating on the component may facilitate mold release after vulcanization. Vaseline can be washed off using soap and water after the rubber ring cures. In general, to help minimize flow turbulence, a tapered transition should be made between the outside diameter of the rod and the inside diameter of the device inlet orifice. For tissue valves, it is likely that the tissue will be damaged in the molding process and should therefore be cut away before molding. A tissue-less valve stent, having all component parts except issue, can facilitate molding of complex shapes. If porous fabric materials are to interface with the silicone rubber, a thin layer of mold release (e.g. Vaseline or wax) should be applied before molding. Experimental devices, (e.g., VSI, BC bi-leaflet tissue valve) can use a plastic rather than a fabric cuff. This simplifies the design of the proto-type valve and fabrication of both the mounting and rubber rings. A molding kit is supplied to assist in making rubber rings. The standard kit consists of the following parts: 1 each, molding jig components 1.8 pounds, GI-1100 Silicone rubber (Silicones, Inc.) 1 each, dispensing syringe (enlarged end port), 35 ml 0.2 pounds, GI-1100 Activator (Silicones, Inc.) 1 each, dispensing syringe, 1 ml 1 each, standard dispensing syringe, 35 ml 1 each, gage tapered tip, #5116TT-B (EFD Inc.) 100 ml, waxed paper mixing cup and wooden spatula 1 ml, mold release grease (Vaseline) in syringe 5.2 GI-1100 Silicone Rubber This rubber is made by Silicones, Inc., P.O. Box 363, 211 Woodbine Street, High Point, NC, 27260, USA. Ph (919) , Fax: (919) It has extremely high tear strength and is a, highly elastic, two-component, tin catalyzed RTV (Room Temperature Vulcanizing) silicone rubber. Specifications include: Tensile Strength (AStm D412) Shrinkage 0.1 % Specific Gravity 1.07 Document No V1.3 ViVitro Labs Inc Page 24 of 118

25 5.3 Ring Moulding Procedure 1. Insert E (dark GREY) is removable. This ring should remain in situ when making rubber rings intended for use in the mitral site of the ViVitro Labs Model Heart and load systems. The Insert E (Dark GREY) should be removed when making rubber rings for use in the Leakage Tester and the HiCycle test systems. This ring may also find use as a filler in the aortic valve mounting site when thin rubber rings intended for use in the mitral position are to be used in the aortic valve mounting site. 2. With the middle ring (YELLOW) over the base (PURPLE), attach the device or replica (light GREY) so that it is centered and square to the base rod (PURPLE). The method of attachment will depend on the device. 3. Fill the assembly with silicone rubber (GI-1100 recommended) which has been deaerated as described later. Prior to complete jig assembly and filling with rubber, it is recommended that rubber first be applied to deep undercut regions using the barrel reservoir and tapered tip (B). This avoids entrapping air in those regions where the flow of the viscous rubber can be problematic. 4. Place the upper ring (GREEN) over the middle ring (YELLOW) and lock with thumbscrew (C). Adjust the locked assembly (GREEN + YELLOW) on the base (PURPLE) so that when rubber fills the cavity, the top level of rubber is even with the top edge of the upper ring (GREEN) and about 2-3 mm of rubber covers the top ridge of the pedestal or device (TAN). This top covering layer of rubber should be sufficient to secure the test device during forward flow conditions. 5. When the rubber is cured, dismantle the molding jig and remove the rubber ring. Remove the upper ring (GREEN) from the middle ring (YELLOW). Two notches on the upper ring (GREEN) allow insertion of a blade type screw driver which can help pry the two rings apart. 6. Push the middle ring (YELLOW) down over the base (PURPLE). 7. Peel and stretch the rubber ring off the device (light GREY). 8. The rubber ring should be trimmed of any excess rubber using small scissors. Trimming should be done carefully to prevent compromising the seal of the ring. 5.4 Preparation of RTV silicon rubber Optional accessories recommended for molding silicone rubber are given below. These items may be obtained from the indicated suppliers. 35 ml/stroke, hand-operated vacuum pump, capable of generating up to 25 inches of mercury vacuum, # P (Cole-Parmer Inc.) 365 ml, Plexiglas vacuum chamber, ¼ inch I.D. tubing, VC9991 air-powered dispenser controller, gas pressure input PSIG; gas pressure output 0-30 ml Barrel reservoir/pistons, #5112CP- NOTE: A standard, hand operated, 35 ml syringe may also be used rather than an air powered dispenser and barrel reservoir/piston combination, however, considerable force of hand is necessary to inject the rubber mixture. The tapered tip (e.g. #5116TT-B) should be attached to the syringe before dispensing the rubber) Document No V1.3 ViVitro Labs Inc Page 25 of 118

26 5.5 Mixing For making one rubber ring, about 15 ml volume of GI-1100 RTV silicone base is required. This volume is best measured and dispensed from the silicone base container using a modified 35 ml syringe which has a large hole in the syringe end. Proceed as follows: 1. Push syringe piston to the end of its travel 2. Insert syringe end into silicone base to a depth of about 2 cm and slowly retract syringe piston to withdraw 15 ml volume. Wipe off excess silicone base from syringe end. 3. Dispense the syringe contents into the mixing cup. 4. Using a 3 ml syringe, dispense 1.5 ml of activator into the cup. 5. Thoroughly stir the mixture with the wooden spatula. Be sure to scrape the sides and bottom of the cup to ensure the correct ratio of base to activator (10:1). Stir slowly until a uniform light blue colour without streaks is achieved. 5.6 De-aerating mixture Immediately after mixing, de-aeration of the mixed rubber to remove entrapped air is always recommended. Removing air bubbles helps assure the rubber rings sealing ability and provides uniformly dense rings. Proceed as follows: 1. Place the mixing cup with the mixed silicone rubber into a small volume vacuum chamber (e.g. part # VC9991, 365 ml chamber volume). 2. Attach a hand operated vacuum pump to the vacuum chamber using a ¼ inch I.D. hose (e.g. # P ). 3. Evacuate to about 24 inches of mercury vacuum. The mixture will expand towards the top edge of the cup and then begin to collapse as the entrapped bubbles burst in the low pressure. The mixture should eventually collapse back to its original volume. The bursting of the bubbles can take several minutes however this time can be reduced by providing multiple evacuation/release cycles during the de-aeration process. During this cycling process, the 24 inches of mercury vacuum condition should be maintained for about 2 minutes. Using this method, the total de-aeration time should be about 10 minutes. 4. Release vacuum and remove the cup from the chamber 5. Pour and spatula the mixture into a 30 ml barrel (e.g. #5112CP-B) and insert piston. 6. Expel barrel air as much as possible. 7. Attach tapered tip (e.g. #5116TT-B) to loaded barrel. 8. Attach the barrel adapter assembly to an air pressure source (e.g. #800V) and set pressure to 40 psig. 9. Slowly inject the mixed silicone rubber into the cavity being careful to avoid the formation of air bubbles or entrapment of air. Allow the rubber to flow around the cavity. The time to fill the cavity with rubber should be about 10 minutes. Allow the rubber to cure (vulcanize) for 16 to 18 hours at 25 C. Lower temperatures and/or low humidity will cause the cure-time to lengthen; conversely, higher temperatures and/or high humidity will cause the cure-time to shorten. Document No V1.3 ViVitro Labs Inc Page 26 of 118

27 6. Pulse Duplicator System Assembly Pulse Duplicator System User Manual The Pulse Duplicator System has several steps to assemble the main mechanical subassemblies, and a few steps for the electrical components installation/configuration. NOTE: It is critical for the assembly steps to be followed in order to assure successful operation. 6.1 Unpacking List of Components Inspect the shipping container for external damage. Retain the shipping carton and packing materials for possible re-shipment. Inspect the system components for damage. If there is evidence of damage contact the carrier, initiate a damage claim, and inform ViVitro Labs by calling or toll free at The following assemblies should be identified on unpacking: 1. SuperPump 2. Ventricle Chamber 3. Atrium Chamber 4. Aortic Chamber 5. Aortic Standoff and and Substitute Probe 6. Compliance Chambers and Tubing 7. Peripheral resistance controller 8. SuperPump Controller 9. Viscoelastic Impedance Adaptor (VIA) 10. Computer 11. Flow Meter and Connector Cables 12. Flow Probe (Not Shown) 13. I/O Module and Connector Cables 14. Ampack, Pressure Transducers, and Connector Cables 15. Molding and Accessories Kit (Not Shown) Document No V1.3 ViVitro Labs Inc Page 27 of 118

28 Optional hardware: Heat Exchanger and Heat Bath Large Compliance Chamber LVOT accessories Transfemoral and Transapical Access Device Digital Manometer The following steps detail procedures for assembling the main components outlined above in the order that they should be installed for a complete unit. 6.2 SuperPump The SuperPump arrives pre-assembled with the following parts: Base Plate Linear Actuator Cylinder Standard Pump Head WARNING: It is recommended that the SuperPump be positioned with its longest dimension horizontal. Should the pump be vertically mounted with the pump head above the motor the possibility of liquid contamination of the mechanical and electrical parts is likely to occur. NOTE: The pump head is made from an electrical non-conductor. In applications where an electromagnetic flow transducer is used this will isolate the path to ground for the transducer from the ground path for the motor. This should improve the signal to noise ratio of the flow signal. Locate the SuperPump on a fixed structure, i.e. a table or frame suitable to accommodate the weight and dimensions of the entire system once put together. The pump head of the SuperPump should extend over the edge of the bench to provide clearance for the ventricle drain assembly. 6.3 Attaching the VIA and the Ventricle Chamber Fasten the VIA directly to the SuperPump using 3x #8-32 x 1 screws and flat washers. Tighten the fasteners evenly and ensure the fasteners are not overtightened. Document No V1.3 ViVitro Labs Inc Page 28 of 118

29 6.4 Attaching the Ventricle Chamber NOTE: The three screws should be tightened evenly and NOT BE OVER-TIGHTENED. Be careful that the bolt entering the blind tapped hole in the Ventricle Chamber does not bottom out when tightened as this will damage the acrylic material of the chamber. Fasten the Ventricle Chamber to the VIA using 3x #8-32 x 1 screws and flat washers. Tighten fasteners evenly and be extremely careful not to overtighten an damage the acrylic. If no VIA is being used with the system. 6.5 Filling the Ventricle Chamber, VIA (if installed) and Pump Head REQUIREMENTS: 0.8 to 1.5 liters of de-aerated fluid. It is further recommended to use distilled water with biocide to reduce bioburden. An appropriate biocide can be determined by a reputable supplier. NOTE: It is recommended that the ventricle chamber be filled with de-aerated fluid to minimize the formation of small air bubbles which may reduce visibility and introduce artifact in echo studies. NOTE: Ensure the SuperPump is set at the home position in the centre of its range movement before filling, therefore creating minimal expansion or collapse of the ventricle membrane during operation. 1. Fill the ventricle chamber with fluid (0.8 liters or 1.5 liters with VIA) via the stopcock. This will fill the pump head, ventricle chamber and VIA (if installed). Fill until the ventricle membrane is slightly compressed. Be sure not to overfill the ventricle chamber which will collapse the ventricle membrane. CLOSE OPEN Relevant stopcock positions Document No V1.3 ViVitro Labs Inc Page 29 of 118

30 2. Close the 2 stopcocks on the VIA (if installed). 3. Remove air by syringe via the stopcock on the ventricle chamber. This involves adding a predetermined amount of fluid and removing an equal amount of air, so that the ventricular membrane is not over or under expanded. Debubble the ventricle chamber and pump head by: attaching a syringe to the stopcock on the ventricle chamber; open the stopcock on the ventricle chamber; if VIA installed, close the two stopcocks on the VIA; elevate the whole system at the ventricle chamber by approximately 30º; debubble/remove the air in the chamber; return to horizontal position when debubbling is completed. Debubble the VIA (if installed) by attaching a syringe to the stopcock closest to the ventricle chamber on the VIA, then: open the stopcock on the VIA; close the remaining stopcocks: one on the ventricle chamber and the other on the VIA; elevate the whole system at the ventricle chamber by approximately 30º; Document No V1.3 ViVitro Labs Inc Page 30 of 118

31 debubble/remove the air in the chamber; return to horizontal position when debubbling is completed. Adjust the fluid volume of the ventricle chamber so that the ventricle membrane is in a relaxed state by: attaching a syringe to the stopcock on the ventricle chamber; open the stopcock; closing the stopcocks on the VIA (if installed); add fluid to the ventricle chamber ensuring the ventricle membrane is in a relaxed state. NOTE: The fluid in the ventricle chamber can remain but should be replaced periodically (<2 months). If the system is used infrequently, drain fluid from the chamber and allow it to dry. 6.6 Accessories Table Installation An accessories table has been supplied in the model left heart crate to accommodate the peripheral resistance and supplementary compliances. Mount the accessories table to the provided flanges on the SuperPump using the two #10-32 x 1 red thumb screws provided. The two threaded holes in the edge of the table for mounting the optional heat exchanger should be at the front left of the SuperPump. Document No V1.3 ViVitro Labs Inc Page 31 of 118

32 6.7 Atrium Chamber Installation NOTE: If you wish to install a mitral flow meter electromagnetic probe you may wish to do this prior to installing the Atrium Chamber (see Installing the mitral flow probe ). 1. Mount the atrium base plate assembly on to the four long stainless steel screw posts situated on the mitral valve side of the mounting block. 2. Tighten the red thumb screws on the assembly to provide a seal around the valve ring. Document No V1.3 ViVitro Labs Inc Page 32 of 118

33 6.8 Aortic Assembly Installation NOTE: If you wish to install an aortic flow meter electromagnetic probe you may wish to do this prior to installing the Aortic Chamber (see Installing the aortic flow probe ). Mount the aortic chamber on the aortic standoff over the ventricular outflow tract flange (over the aortic valve). Tighten the red headed thumbscrews. NOTE: When positioned correctly, the side of the aortic assembly with the three ports colour coded RED, YELLOW, and BLUE should be facing towards the accessories table. NOTE: The aorta viewing tube and window assembly is adjustable and is locked into position using three bolts. The viewing tube window should never be closer than 20 mm from the end of the aorta outlet tube. Document No V1.3 ViVitro Labs Inc Page 33 of 118

34 6.9 Flow Probe(s) Initial Operational Check Components: Flow Meter Flow Probe PHONO to BNC Cable Before installing the flow probe(s), an initial operation check of the flow measurement system should be performed as follows. NOTE: This should be done for each flow probe and its accompanying flow meter. 1. Check that the voltage switch on the back panel of the unit is set for your mains supply (115 or 230 VAC). If it must be switched, ensure the correct fuses are installed in the meter as per the instructions on the rear of the unit. If it is necessary to change the power plug style then the following conductor colours apply: black=phase, white=neutral 2. Check POWER OFF (down) and PROBE switch OFF 3. Plug the power cord into the mains supply. NOTE: The Flow meter must be properly grounded to the probe site. 4. Connect PULS output on the back panel to the INPUT CH. 4 on the I/O Module. 5. Place the flow probe and ground lead into a non-metallic container filled with a conductive fluid such as saline. (It does not function in air or pure water.) 6. Insert the ground lead cable into the flow meter ground jack on the front panel of the meter. 7. Connect the 8-pin probe plug to the flow meter jack with the correct registration as indicated by the missing pin. 8. Set the MEAN switch to HI. 9. Set the outer dial on the RANGE switch to give a PFX (probe factor multiplier) of The Hz RESPONSE and PROBE FACTOR controls should be left as set during ViVitro Labs Inc calibration as shown on the calibration sheet. 11. Turn the POWER switch on. 12. A switch transient may cause the ALARM indicator to illuminate. Depress the ALARM indicator to reset the meter. Allow ten minutes for the instrument to stabilize. NOTE: The green READY light should remain on when the meter is measuring flow. Document No V1.3 ViVitro Labs Inc Page 34 of 118

35 13. With the PROBE switch set to OFF adjust the ZERO control to give a display meter reading of zero. The ZERO control is a 15-turn potentiometer and normal setting would be approximately mid-position, 7 ½ turns clockwise from the extreme anti-clockwise rotation. NOTE: ZERO control is functional with or without the flow probe attached and the no-signal level of the back panel MEAN and PULS outputs are set by the ZERO control. The withsignal level should be approximately the same as the no-signal level under static flow condition. 14. With the PROBE switch set to NULL adjust the NULL control until the display meter indicates a MINIMUM reading. 15. With the PROBE switch set to BALANCE rotate the BALANCE control so that the meter reads approximately zero. This position should be close to that set by ViVitro Labs Inc during pre-shipment calibration. 16. With the PROBE switch set to either + or - (Normal placement of the probe, dictates flow is in the + direction) If necessary, use the ZERO control until the meter shows zero. NOTE: If the meter is off scale, check for improper grounding or air bubbles in the probe lumen which can be removed using a cotton swab. Alternate + and while adjusting Balance for least difference. 17. Move the probe so that an oscillatory flow of saline moves through the probe lumen (caused by moving the hand or implement in the fluid manually). The display meter should show an oscillatory signal above and below zero. The back panel PULS output should also follow this signal. This may be verified on a flow monitor or via the ViViTest software. Turn the PROBE switch OFF when flow is not being monitored. Successful completion of the above steps indicates a functioning flow meter system and mounting of the flow probe into the Pulse Duplicator System can proceed. Once mounted in Pulse Duplicator System, step 5 and 17 are not applicable. Document No V1.3 ViVitro Labs Inc Page 35 of 118

36 6.10 Installation of Flow Probes There are two positions on the Pulse Duplicator System where electromagnetic probes may be installed in the place of substitute probes: in the aortic site and the mitral site AORTIC MITRAL When a single probe/flow meter is used, the displacement of the pump is differentiated over time to obtain the total flow into the system, and then the measured flow from the site with the sensor is subtracted from this number to obtain the flow in the site without a sensor. In every case, a ground connection between the flow meter and the probe site is necessary. SYNCH on the back panel of the flow meter is used only when two flow meters are connected Installing the Aortic Flow Probe The aortic probe is located between the ventricle chamber and the aortic chamber on the aortic standoff. 1. Remove the aortic assembly (loosen thumb screws and lift off tapered screw posts. 2. Unscrew the four tapered screw posts to remove the white mounting on the aortic standoff. This will reveal a further 4 screws to remove the remaining structure. Remove the four screws to unclamp the substitute flow transducer (white Delrin spacer). 3. Remove substitute flow probe. 4. Insert electromagnetic flow probe using the supplied flow channel alignment jig to assist in centering the flow probe between the aortic housing clamp, ventricular outflow tract and valve mounting base. Take care to align the flats on the alignment jig with the probe electrodes to prevent marring the electrodes. 5. Replace and evenly tighten the four bolts. Do not use excessive torque. 6. Replace and adjust the four tapered studs so that when the thumbscrews are being tightened onto the tapered surface of these studs, the mating flanges pull together and provide a seal around the silicone ring. Document No V1.3 ViVitro Labs Inc Page 36 of 118

37 6.12 Installing the Mitral Flow Probe The mitral probe is located between the ventricle chamber and the atrium chamber. 1. Remove the left atrium assembly (loosen the four thumbscrews and lift). 2. Loosen the four bolts securing the mitral flow channel to the atrium chamber. 3. Remove white Delrin spacer. 4. Insert electromagnetic flow probe. Carefully align probe to minimize turbulence. 5. Tighten evenly the four bolts. Do not use excessive torque Aortic Compliance Chamber Installation 1. Mount the Aortic Compliance Chamber on to the Accessories Table using two red #8-32 x ¾ thumb screws provided. 2. Connect the silicone tube from the top of the compliance chamber to the Aortic Chamber at the marked YELLOW outlets/inlets Aortic Room Compliance Chamber 1. Mount the Aortic Root Compliance Chamber on to the Accessories Table using two #8-32 x ¾ red thumb screws provided. 2. Connect the silicone tube from the top of the compliance chamber to the Aortic Chamber at the marked BLUE outlets/inlets. Document No V1.3 ViVitro Labs Inc Page 37 of 118

38 6.15 Peripheral Resistance Controller 1. Mount the Peripheral Resistance Controller on the Accessories Table using the two #10-32 x 1 ¼ screws provided. 2. Connect the silicone tube from the outlet (marked GREEN) of the controller to the inlet of the Atrium Chamber (marked GREEN). Connections with no Heat Exchanger 3. If no heat exchanger is being used, connect the silicone tube from the RED outlet of the Aortic Chamber to the RED inlet of the controller. If using a heat exchanger, proceed to Section Connections with a Heat Exchanger Document No V1.3 ViVitro Labs Inc Page 38 of 118

39 6.16 Heat Exchanger System Installation (Optional) Components: Heat Exchanger with Mounting Flange Heat Bath 1. Mount the Heat Exchanger to the side of the Accessories Table (where screw holes are located). 2. Connect the silicone tube from the Test Fluid Outlet (top) of the Heat Exchanger to the Peripheral Resistance Controller. 3. Connect the silicone tube from the outlet of the Aortic Chamber marked RED to the Test Fluid Inlet on the Heat Exchanger (under side). 4. Connect the silicone tube from the right inlet on the Heat Exchanger to the outlet on the Heat Bath. 5. Connect the silicone tube from the left outlet on the Heat Exchanger to the inlet on the Heat Bath. 6. Fill the Heat Bath with fluid. NOTE: It is advised to add an algaecide to prevent algae growth. 7. Turn the Heat Bath on and set to desired temperature (i.e. 37.0ºC) Silicon Hose Installation Confirm that the four colour coded silicone rubber hoses are connected correctly between colour coded ports: 1. YELLOW: from the port on the top of the aortic assembly to the smaller of the two compliance bottles. This provides characteristic compliance supplementary to that in the aortic assembly. 2. BLUE: from the port to the air volume surrounding the aortic root to the larger of the two compliance bottles. This provides supplementary aortic root compliance. 3. RED: from the outlet port in the aortic root enclosure to the inlet of the peripheral resistance (or the heat exchanger inflow port.) 4. GREEN: from the outlet of the peripheral resistance to the inlet of the left atrium assembly Document No V1.3 ViVitro Labs Inc Page 39 of 118

40 6.18 Electrical Connections The 6 electrical components used to control and monitor the mechanical components of the Pulse Duplicator System are: 1. SuperPump 2. SuperPump Amplifier 3. Flow Meter 4. AmPack 5. Computer 6. I/O Module See Page 42 for an annotated image of the components. STOP: CHECK YOUR COUNTRY S POWER SUPPLY AMPERAGE AND VOLTAGE PRIOR TO CONNECTING INTO A POWER SUPPLY ViVitro Labs Inc. will endeavor to have your system s components adapted to meet your country s power supply, however, as an extra precaution, it is advised that all equipment designated to be connected to a power supply be checked prior to use. 1. Check that the voltage setting on the back panel of the Flow meter and AmPack are set for your supply voltage BEFORE connecting to the power supply. Ensure that the operating voltage of the Heat Bath is correct for your region before connecting to the power supply. 2. Where the voltage setting is incorrect adjust the setting manually according to the hardware s instructions. 3. Where it is necessary to adapt the power supply plugs the following line cord colours should be observed. black=phase, white=neutral, green=ground. The diagram on the following page shows a schematic detailing the electrical connections that need to be made to make the system operational. The following table details these connections: Diagram Colour Hardware From Label Hardware To Label Reference Red AM Pack Bottom Left Aortic Chamber Aortic Transducer Dark Red AM Pack Bottom Right I/O Module CH 0 (INPUT) Yellow AM Pack Middle Left Ventricle Chamber Ventricular Transducer Orange AM Pack Middle Right I/O Module CH 1 (INPUT) Sky Blue AM Pack Top Left Atrium Chamber Atrial Transducer Dark Blue AM Pack Top Right I/O Module CH 2 (INPUT) Green SuperPump Amplifier DL/DT OUTPUT I/O Module CH 3 (INPUT) Navy Blue Flow Meter PULSE I/O Module CH 4 (INPUT) Green Flow Meter Probe Cable Aortic or Mitral Flow Probe Flow Probe location Black Flow Meter Ground Aortic or Mitral Grounding Pin Standoff Grey SuperPump Amplifier POSITION OUTPUT I/O Module CH 5 Brown SuperPump Amplifier DRIVE INPUT I/O Module CH 0 (OUTPUT) Black SuperPump Amplifier LINEAR ACTUATOR SuperPump Motor Green I/O Module USB Computer USB Document No V1.3 ViVitro Labs Inc Page 40 of 118

41 6.19 Electrical Connection Diagram Electrical connections for the Pulse Duplicator System Document No V1.3 ViVitro Labs Inc Page 41 of 118

42 7. Testing Set-up Procedures Pulse Duplicator System User Manual The following sections describe the steps required to prepare an assembled system for testing. 7.1 Initial Assembly Procedure Refer to Section 6 to ensure the systems initial assembly is complete before continuing. Going forward it is assumed that all the hardware and electrical components have been connected. The following sections provide the user with the procedures for set-up of the Pulse Duplicator System for testing. These procedures provide operation for ideal physiological conditions known to ViVitro Labs. The diagram below provides an illustration of the fundamental set-up, and operational depiction. 1. SuperPump 2. Ventricle Chamber 3. Atrium Chamber 4. Aortic Chamber 5. Aortic Standoff and and Substitute Probe 6. Compliance Chambers and Tubing 7. Peripheral resistance controller 8. SuperPump Controller 9. Viscoelastic Impedance Adaptor (VIA) 10. Computer 11. Flow Meter and Connector Cables 12. Flow Probe (Not Shown) 13. I/O Module and Connector Cables 14. Ampack, Pressure Transducers, and Connector Cables The Standard Pulse Duplicator System. A complete list of components for each assembly can be found within the Appendices. Document No V1.3 ViVitro Labs Inc Page 42 of 118

43 7.2 Installation of Valves NOTE: A silicon rubber ring around the valve ring or sewing ring should be used for mounting valves in the Pulse Duplicator System. This ring also provides the seal between the atrium chamber and the mitral valve on the ventricle chamber mounting block and between the aortic valve mounting flange and the aortic standoff. The soft rubber also helps simulate compliant tissue. The valves in their rubber rings can be inserted into the valve mounting flanges. The mitral valve corresponds to the site with the longer tapered studs in the valve mounting block. The aortic valve mounts in the aortic outflow tract downstream of the aortic flow transducer or substitute Delrin ring. Note that the valves in their rings can be rotated around their stream wise axis and mounted in different orientations. The mitral and aortic valves are retained by the atrium and aortic standoff, respectively. 7.3 Priming / Preparing the Chambers 1. Add the test fluid through the atrium chamber, located above the mitral valve 2. The Aortic, Ventricle and Atrium Chambers will fill with fluid. Continue until fluid is near the top of the aortic outflow in the Aortic Chamber. 7.4 Air Bubble Removal To ensure the data you acquire is accurate, it is important to debubble the chambers prior to running your tests. Instructions: 1. Install a catheter line to the stopcock at the top of the aortic chamber and place the end of the catheter in the atrium chamber to collect overflow 2. Ensure the lid is fastened to the top of the aortic chamber 3. Check that the peripheral resistance controller is open at least two full turns 4. Via the Amplifier: a. Start the SuperPump by slowly turning the Amplitude knob b. Adjust the Amplitude setting to a low stroke volume (approx. 30 ml/stroke) 5. Open the stopcock to vent air from the inside of the aortic chamber. Document No V1.3 ViVitro Labs Inc Page 43 of 118

44 6. Add fluid to the atrium as necessary to the system to prevent air bubbles from being circulated. 7. Close the stopcock when all air has been displaced by test fluid NOTE: It may be necessary to increase the pumping rate/stroke volume and reduce the Peripheral Resistance to remove all air bubbles. Monitor aortic pressure and avoid excess pressure in the system. 7.5 Characteristic Compliance and Liquid Level Adjustment Note: The instructions for Aortic Root Compliance Chamber and Aortic Compliance Chamber fluid levels should be conducted simultaneously. Aortic Root Compliance 1. With the stroke volume set at a given value (e.g beats/minute) the pressure of the air above the fluid surrounding the Aortic Root should be adjusted so that the highest level of fluid in the aortic housing almost reaches, but does not occlude the tube port making connection to the Aortic Root Compliance Chamber. 2. Using a syringe apply or release air as needed to the Aortic Root Compliance Chamber by way of the luer fitting connected to the tube at the compliance chamber. NOTE: If fluid enters the aortic root tube it will alter the Aortic Pressure waveform. This will be noticed by a dip in the Aortic Pressure waveform after the Ventricle Pressure drops. Result: Approximate air volumes will be: Aortic Root Chamber 35 ml plus Aortic Root Compliance Chamber (incl. hose) 615 ml Totaling 640 ml If desired, this volume and hence Aortic Root Compliance can be reduced by adding water to the Aortic Root Compliance Chamber to reduce the air volume. Aortic Compliance The fluid in the Aortic Chamber should be allowed to rise approximately 2.5 cm. 1. Using a syringe apply or release air as needed to the Aortic Compliance Chamber by way of luer fitting connected to the tube at the compliance chamber. Note: During collection of low cardiac outputs (e.g. 2 LPM C.O.), it may be necessary to clamp the Aortic Compliance Chamber hose to reduce the compliance, see comments in Pressure Waveform Adjustment section. Result: Approximate air volumes will be: Aortic Chamber 300ml plus Aortic Root Compliance Chamber (incl. hose) 350ml Totaling 650ml If desired, this volume and hence Aortic Root Compliance can be reduced by adding water to the Aortic Root Compliance Chamber to reduce the air volume. Document No V1.3 ViVitro Labs Inc Page 44 of 118

45 7.6 Peripheral Resistance Adjustment The peripheral resistance can be adjusted to regulate aortic pressure. This will also affect cardiac output. NOTE: The peripheral resistance adjustment will also influence the pressure throughout the system so it is recommended to make incremental adjustments while monitoring the air level of the compliance chambers and mean aortic pressure. Care should be taken NOT to occlude the Peripheral Resistance or connection tubing completely. This would rapidly cause excess pressure. Adjustment of the Peripheral Resistance may also change the Aortic Root external fluid level. This level should be adjusted as shown in Section Rotate the peripheral resistance outer housing clockwise to increase its resistance to flow. The vernier scale can be used as a guide for repeatability. 2. Monitor the mean Aortic Pressure on the ViViTest software while increasing the stroke volume to ensure that you do not over pressurize the system (for concise instructions see ViViTest Software Operating Manual). 3. During normal operation, the peripheral resistance should be adjusted so that this pressure ranges between 80 and 120 mmhg. 7.7 Pressure Waveform Adjustment The shape of the Aortic and Ventricle Pressure waveforms is influenced by the Aortic Root and Aortic Compliance Chambers. Additionally, the Viscoelastic Impedance Adapter (VIA) may influence these waveforms. In particular, reduction of the Compliance Chamber pressure will increase the ratio of maximum to minimum Aortic Pressure. At low stroke volumes (e.g. 35 ml/stroke ~2 LPM C.O.) elimination of this compliance may give Aortic Pressure wave shapes closer to physiologic ones. Normally, the Aortic Root Compliance Chamber air volume is left at its maximum value of 640 ml. Instructions: 1. The tubing to the air compliance chambers can be occluded by closing the plastic clamps. These clamps can be closed just prior to reducing pump stroke volume to zero to prevent air bubbles from entering the test fluid flow loop. 2. You may also release air from the Aortic Root Compliance Chamber when slowly turning down the stroke volume to maintain the proper water levels. This helps to stay prepared for continuous collections. Document No V1.3 ViVitro Labs Inc Page 45 of 118

46 7.8 Viscoelastic Imedance Adapter (VIA) adjustments The VIA will increase ventricle compliance and reduce the rate of increase of ventricle pressure during ventricle contraction. The VIA action consists of the resistance element and two air compliance chambers. Vary the volume of air in the output chamber and source chamber to attain the desired pressure waveform. 7.9 Mean Atrial Pressure The Mean Atrial Pressure value can be adjusted by altering the fluid level in the Atrium Chamber. There should be cm of fluid above the mitral valve Transducer Settings Four ports with stopcocks are provided for the insertion of pressure transducers to measure Aortic, Aortic outflow tract, Ventricle, and Atrial pressures. Instruction: Insert the pressure transducers directly into the stopcocks at the pressure measuring ports. Care should be taken to remove air bubbles in the fluid line from pressure transducers by flushing the line using a syringe (distilled water is recommended). Attach the syringe to the outer luer fitting, open inner and outer fitting, and slowly pull fluid through transducer. Repeat until all bubbles are removed and away from transducer sensor. Apply atmospheric pressure to the pressure transducers by turning the appropriate stopcocks to the closed position (this allows the transducer to measure air/atmospheric pressure). NOTE: Excessive tightening of the Luer lock cap should be avoided. Pressure transducers may be damaged by over-pressure. This can occur if a finger occludes the transducer pressure port during transducer flushing. RAPID STOPCOCK CLOSURE may also produce excess pressure. Please contact ViVitro Labs Inc. regarding use of other pressure transducers with the ViVitro Labs Inc. Model AM9991 amplifiers (e.g. Millar catheter tip MPC-500). NOTE: Atmospheric pressure is useful as a zero pressure reference. The pressure on the transducer sensing element will be atmospheric plus or minus the static head. This includes fluid in the stopcock and in the pressure transducer. If this static head and atmospheric pressure do not change (i.e. orientation of transducer and atmospheric pressure are constant) then the reference pressure will be constant for each transducer. NOTE: With no flow of test fluid, the pressure transducers will record the static head of pressure on the transducer sensing element. This will differ from the static wall pressure by an amount equivalent to the head level difference between wall pressure measuring point and the sensing element. To compute transvalvular pressure under pulsatile flow conditions, compensation for the static head of pressure should be made. This compensates the pressure transducers to a zero offset for differential measurement, referenced as compensated pressure. Document No V1.3 ViVitro Labs Inc Page 46 of 118

47 Experimental Measurement: Experimental measurement of the compensation to be applied can be made with the same fluid head in the model above all pressure measuring ports. The ViViTest software handles experimental determination of the static head pressures as part of the pressure calibration routine. Alternatively, these static head pressures may be manually input into the software. When the test fluid is saline, the approximate values to be input are -6.5 mmhg for the aortic pressure site and -0.2 mmhg for the straight left atrium or -3.5 for the angled/rectilinear left atrium site. These values are referenced to the ventricle pressure measurement site, thus the ventricular pressure offset will be 0.0. Document No V1.3 ViVitro Labs Inc Page 47 of 118

48 8. System Preparation for Calibration Pulse Duplicator System User Manual Verify power is On for all instruments. Verify hardware and fluids: o Pump head (and VIA optional component) are fluid filled and debubbled; o Flow meter probe is in place and connected; o Atrium, Ventricle (silicone ventricular membrane is in a static state), Aortic Root and Aorta Chambers are fluid filled according to their specifications and debubbled; o Compliance Chambers are compliant; o Transducers are in place and connected; o Peripheral Resistance is connected; o Heat exchanger is connected (optional component). Verify electrical components and connections: o Pump controller is turned on and desired waveform control is selected. Ensure Limit is not illuminated (if it is, switch is moved in the direction of the arrow). o Flow meter turned on, MEAN is set to HI, ground cable is connected to Flow Probe pin. If the Alarm is illuminated depress and allow 10 minutes to stabilize. It is recommended to set the Range to 100 and Hz Response to 30Hz. o Pressure Amplifier turned on. Low pass filter settings set to recommended 30Hz. o Data Acquisition system turned on and ViViTest software loaded and running. 8.1 Pump Calibration Operational Check Verify the Pump Controller is properly connected to all cable connections. The power supply is auto-detectable for 230/50Hz or 110/60Hz. Switch power button located on the back panel to ON. Piston should immediately home to the central cylinder position. The LCD screen will display any errors and provide status of the controller. Using the waveform button located on the front panel, select the desired waveform. If using ViViTest, the external channel should be selected. Note: For further pump control features - please see the SuperPump AR series user manual. NOTE: Avoid large piston strokes that could activate the pump limit switches. Note that if the push or pull limits of the pump are reached, the pump will automatically reset to the home position. 1 Pump Piston Transducer Calibration The pump piston linear displacement is measured for each unit. The factor in mv/mm is provided by ViVitro manufacturing. Follow the ViViTest pump calibration step to ensure the correct Pump Calibration Factor is entered. 8.2 Drive Waveform Generator Operational Check The Pulse Duplicator is operated by unique complex analog waveforms supplied by the analog output of the I/O Module. See the ViViTest User Manual for instructions. NOTE: Do not change the waveform setting while the pump is in operation. Set amplitude to zero before changing the waveform. The selected waveform will dictate the operation of the pump to a prescribed push/pull (systole/diastole) ratio corresponding to a systolic duration percentage, characteristic waveform, and beat rate in beats per minute. Document No V1.3 ViVitro Labs Inc Page 48 of 118

49 8.3 Calibration of Flow Probe Methods There are two distinctive methods to collecting flow measurements. Each should be considered prior to starting, so that the proper set-up and recognition by the ViViTest software is performed. 1. Direct Flow This is the most common type of measurement. The flow probe should be mounted in the position of interest, i.e. aortic valve flow measurement requires the flow probe to be mounted on the Aortic Chamber side of the Heart Model. The ViViTest software will automatically determine flow for the probe site based on the target probe site and target valve. If these entries are the same the software will recognize the system as configured for direct flow measurements. 2. Derived Flow This type of measurement is used when the experimentalist notes a problem that is noticeable as leakage flow computing to a positive value. This can result when using the electromagnetic flow measuring system and a disturbance of the flow field around the flow probe occurs. The problem can be particularly noticeable when using mechanical valves where small leakage paths can give rise to relatively high velocity regurgitant fluid jets and consequent highly disturbed flow in the model flow channels. (Positive leakage can also be caused by excessive valve compliance during back pressure and modifications to the valve mounting should be considered as the first source to resolve the problem; derived flow is best used when no other solution is available to the investigator). The derived flow method calculates the flow for the target valve by placing the flow probe and supplied competent valve (i.e. no leakage) in the companion site and computing the flow from the dl/dt pump signal. Given the check valve is designed not to leak the ventricular flow or pump displacement can be assigned as the target valve flow. NOTE: The use of derived flow has been validated by comparing derived flow leakage rates with those obtained from leakage column measurements. This validation was made using a ViVitro Labs Pulse Duplicator System with left atrium and mitral flow channels in line. The method is only valid with NO compliance. The VIA and any air in the hydraulic chamber must be removed. The competent valve must be used in the companion site and the rectilinear atrium chamber. This method is not recommended for the mitral position or with vertical atrium chambers. NOTE: The ViViTest software alternately uses the same prompts for Direct or Derived flow measurements. The ViViTest will provide flow measurements for both the aortic or mitral position regardless of the configuration, so your choice of target probe site and target valve determines if the measurement for the target valve is direct or derived. Document No V1.3 ViVitro Labs Inc Page 49 of 118

50 8.4 Flow Meter Operational Check NOTE: Warning: Incorrect mains voltage setting may cause damage to the flow meter Installation Instructions: Refer to the Flow Meter Manual before changing the power plug style. Check that the voltage switch on the back panel of the unit is set for your mains supply (115 or 230 VAC). With POWER OFF (down) and PROBE switch OFF plug the power cord into the mains supply. NOTE: The Flow Meter must be properly grounded. Connect PULS output on the back panel to the flow-monitoring device. A PHONO to BNC cable for connection to the I/O Module is supplied. Connect the 8-pin probe plug to the Flow Meter jack with the correct registration as indicated by the missing pin. The flow probe requires a conductive fluid such as saline to properly function. It will not function in air or pure water. Connect the ground jack on the front panel of the Flow Meter to the test fluid ground point corresponding to the flow probe site. Set the MEAN switch to HI. Set the outer dial on the RANGE switch to give a PFX (probe factor multiplier) of 1. The frequency response recommendation is 30Hz. The PROBE FACTOR should be set to the manufacturer s recommendation. See label below. The probe factor, shown in the red circle, translates to a setting of 4.38 on the flow meter potentiometer. Reference the probe factor calibration label provided with the flow probe for proper set-up. Following the ViViTest software instructions for flow calibration, the software will automatically adjust create a calibration factor for the flow meter signal to equal the pump dl/dt. If a significant drift is noticed in the flow meter conversion factor, the flow meter and probe may require calibration. Turn the POWER switch on. A switch transient may cause the ALARM indicator to illuminate. Depress the ALARM indicator to reset the meter. Allow ten minutes for the instrument to stabilize. The green READY light should remain on when the meter is measuring flow. With the PROBE switch set to OFF: Adjust the ZERO control to give a display meter a reading of zero. NOTE: The ZERO control is functional with or without the flow probe attached and the no-signal level of the back panel MEAN and PULS outputs are set by the ZERO control. The with-signal level should be approximately the same as the no-signal level under static flow condition. Document No V1.3 ViVitro Labs Inc Page 50 of 118

51 8.5 Null, Balance and Zero NOTE: The following MUST be performed with the system filled with 0.9% NaCl (saline solution). With the PROBE switch set to OFF, adjust the ZERO control until the display meter reads zero. Set the PROBE switch set to NULL. Adjust the NULL control until the display meter indicates a MINIMUM reading. Set the PROBE switch set to BALANCE. Rotate the BALANCE control so that the meter reads zero. This position should be close to that set by ViVitro Labs Inc during preshipment calibration. Set the PROBE switch set to either + or (Normal placement of the probe, dictates flow is in the + direction) If necessary, use the BALANCE control until the meter shows zero. NOTE: If the meter is off scale, check for improper grounding or air bubbles in the probe lumen which can be removed using a cotton swab. To verify the flow meter is set and/or operating correctly, use the ViViTest software flow calibration screen to verify an oscillatory flow when the pump is operating. Reference the flow meter manual if the flow meter is suspected of not functioning properly. Turn the PROBE switch OFF when flow is not being monitored or NO test fluid is present. NOTE: Warning: Probe can be damaged if left on when NO test fluid is present. Calibration of the Flow Meter may now be performed using the ViViTest software. 8.6 System Check The ground jack on the front panel of the Flow Meter should be connected to the test fluid ground point corresponding to the flow probe site (see cabling schematic). If the fluid ground point is changed, the Flow Meter should be re-adjusted per steps starting at the NULL step. When the Flow Meter is supplied by ViVitro Labs Inc, calibration of the unit using the ViViTest software is carried out prior to shipment. The calibration applies ONLY when the front panel control settings are those on the calibration sheet. The calibration sheet gives the calibration factor in units of mv/(ml/s). This may be used for manual input to the ViViTest software, however it is recommended that the flow meter be calibrated daily. The calibration sheet compares the volumetric flow rate generated by a piston pump and the resulting flow meter signal. During calibration of the flow transducer no air must be in the VIA. The ViViTest software will equalize the flow meter output to the instantaneous net ventricle volumetric flow rate generated by SuperPump using a sinusoidal waveform at 70 or 100bpm. This volumetric flow rate is obtained by using the piston displacement signal differentiated with respect to time. This signal is output from the SuperPump controller back panel through the output labeled dl/dt. The dynamic flow transducer calibration is performed with no valves in the Model Heart. To take the place of disc valves, the accessories kit contains a silicon rubber flow straightener sealing ring and also a solid seal. The solid seal assures zero flow in the occluded site. NOTE: After filling/refilling with test fluid, the meter should be re-adjusted as outlined in steps starting from NULL (4.3.2) Document No V1.3 ViVitro Labs Inc Page 51 of 118

52 8.7 Calibration Procedure for Aortic Flow Probe The diagram below illustrates the proper set-up for Aortic Flow calibration. Stoppers may be used as shown in the diagram or the tubing can be clamped. If the stoppers are used care should be taken to remove the tubing without damaging or breaking the acrylic hose barbs. It is recommended that the flow probe be calibrated using pulsatile flow generated in the model. This can readily be done using the SuperPump with no air in the hydraulic chamber and VIA. The instantaneous net ventricle volumetric flow rate should equal the flow rate generated by the SuperPump. The ViViTest software will facilitate the sequence of steps to determine these measurements and determine the calibration factor. A sine wave from bpm is required to create a stable oscillating flow. The previously calibrated pump volume dl/dt signal is matched to the flow probe signal. To proceed with aortic flow probe calibration: 1. Insert probe in aortic site as described previously; 2. Remove window assemblage on top of aortic chamber (loosen bolts holding three retaining tabs, rotate tabs to window release position, and pull up on window; Document No V1.3 ViVitro Labs Inc Page 52 of 118

53 3. Occlude RED and BLUE ports on aorta housing block with rubber stoppers or clamp the tubes securely and clamp drain hose; 4. Place silicon rubber annular seal in aortic site and secure the aortic chamber with thumbscrews; 5. Occlude the mitral site with solid silicon rubber seal; 6. Place atrium assembly over solid seal and secure by tightening the four thumbscrews; 7. Add fluid (must contain 0.9% NaCl) to load reservoir until level just reaches mitral site. Continue to fill the system through the open top on aortic chamber reservoir until the test fluid level reaches the level indicated in the diagram on above; 8. Remove all entrapped air through ports occluded by stopper 1 & 2 (may be necessary to tilt model). 9. It is important to remove all air from the aortic root assembly. This can be done via the BLUE port. It may be necessary to tilt the model so that all air can exit through this port. Switch on the drive to SuperPump and record the piston displacement, piston displacement differentiated with respect to time (dl/dt), and flow transducer signals. Using the ViViTest software following the FLOW Calibration steps. The phasing between these signals can also be determined. If the electronic filters on the instruments are matched (typically, either 30 Hz or 100 Hz low pass filters) then any measurable phase difference for these signals is likely due to the characteristics of the transmission path between the flow generator (piston) and the flow probe. NOTE: Always verify the flow zero, reset electronically by following the software prompts for rezero. 10. Turn the pump controller amplitude to the desired pump stroke volume; typically 70ml. 11. Verify the proper flow meter PROBE FACTOR value obtained from the manufacturer calibration label is set; 12. The system is now ready for flow calibration in ViViTest. Document No V1.3 ViVitro Labs Inc Page 53 of 118

54 8.8 Calibration Preparation of Mitral Flow Probe The diagram below illustrates the proper set-up for Mitral Flow calibration. Stoppers may be used as shown in the diagram or the tubing can be clamped. If the stoppers are used care should be taken to remove the tubing without damaging or breaking the acrylic hose barbs. Mitral flow calibration is conducted with the same principals as those previously described for aortic flow calibration. Similarly, set-up the model with no air in the pump head and VIA. The instantaneous net ventricle volumetric flow rate should equal the flow rate generated by SuperPump. This flow rate is obtained by using the piston displacement signal differentiated with respect to time. This is output from the SuperPump power amplifier back panel through the output labeled dl/dt. The dynamic flow probe calibration is performed with no valves in the system. To take the place disc valves, mounting rings from the accessories kit contains a silicon rubber flow straightener sealing ring and also a solid seal for the aortic valve non-test site. To proceed with mitral flow probe calibration preparation: 1. Insert flow probe in mitral site as described in the installation section; 2. Place silicon rubber annular seal in mitral site; 3. Place atrium assembly over seal and secure by tightening the four thumbscrews; Document No V1.3 ViVitro Labs Inc Page 54 of 118

55 4. Use black rubber stopper to occlude the green hose port or clamp securely and clamp drain hose; 5. Add fluid (must be 0.9% NaCl) to the atrium reservoir until fluid reaches aortic valve site; 6. Occlude aortic site with solid silicon rubber seal without trapping air beneath seal; 7. Place load assembly over solid silicon rubber seal and secure by tightening thumbscrews; 8. Complete filling of chamber until the test fluid level reaches the level indicated in the diagram above; 9. Switch on the drive to SuperPump and record the piston displacement, piston displacement differentiated with respect to time (dl/dt), and flow probe signals. Using the ViViTest FLOW Calibration steps, the calibration of these signals can be conveniently determined. The phasing between these signals can also be determined. If the electronic filters on the instruments are matched (typically, either 30 Hz or 100 Hz low pass filters) then any measurable phase difference for these signals is likely due to the characteristics of the transmission path between the flow generator (piston) and the flow probe. 10. Turn the pump controller amplitude to the desired pump stroke volume; typically 70ml; NOTE: Always verify the flow zero, reset electronically by following the software prompts for rezero. 11. Verify the proper flow meter PROBE FACTOR value obtained from the manufacturer calibration label is set; 12. The system is now ready for flow calibration in ViViTest. A CAUTION about FLOW Measurement Electromagnetic flow probes (e.m.probes) are best suited to measurement of uniform streamline flow (non-turbulent). Flow in Model Heart channels may deviate considerably from ideal for e.m. probes, particularly during the leakage flow phase when regurgitant jets from valves produce unfavorable flows in the probe lumen. This can be most problematic at high cycle rates and flows. The flow signal must therefore be interpreted advisedly. In particular, a positive flow signal seen during the leakage phase for a valve known to seal is indicative of an erroneous flow signal. This problem may be reduced by rotating probe (or valve) about the central flow streamline to minimize direct impingement of jets on the electrodes. Additionally, a rigid or less compliance mount can reduce the effects of positive leakage. Another solution for this flow measurement problem is to use indirect flow measurement. For a leaking valve in the aortic test site, the alternative method of measuring flow is by placing the flow probe in the mitral site and eliminating any compliance between pump and ventricle. In this way, the pump displacement differentiated with respect to time (dl/dt) becomes the ventricle net volumetric flow rate. In this case, with the e.m. probe in the mitral site, impingement of jets on electrodes should be less problematic because of the mitral flow channel configuration. The ViViTest software handles synthesis of the derived aortic flow signal from the e.m. probe mitral site flow signal and the pump dl/dt. Document No V1.3 ViVitro Labs Inc Page 55 of 118

56 8.9 Calibration of Pressure Measurement System (AP9991) Initial Setup 1. Switch the unit ON (rear panel). Allow the amplifiers to warm up for 30 minutes to achieve minimum drift. NOTE: Warm up time can be avoided by leaving the unit on continuously. This will not damage the system. Select the desired filters using the filter switch located on the front panel of each amplifier. For pulse duplicator measurements 30 Hz is recommended. o INPUT: always use the locking screws on the cable connector. o GAIN: adjustable by 15 turn potentiometer. o FILTERS: A five position switch selects upper frequency cut-off. Normally this should be lower than the natural frequency of the transducer. o SHIFT: 15-turn potentiometer; each turn shifts output 2.5 volts between full scale ±12 volts. For additional specifications or instructions refer to the Pressure Measurement User Manual. GAIN & SHIFT Adjustment and Calibration Each amplifier module and pressure transducer may be calibrated following the procedure given below. 1. Using a Manometer, an appropriate range and accuracy, and a volt meter with a display of and volts DC to an accuracy greater than the pressure gauge accuracy. Follow the ViViTest software calibration steps, inputting range (i.e mmHg), recognition of atmospheric pressure voltage (-6.0 volts; adjust SHIFT as needed), and recognition full scale voltage (6.0 volts; adjust GAIN as needed). NOTE: To avoid poor signal to noise ratio and recording of erroneous data, the full scale capability of recording instruments should be matched to the range of the dynamic signals to be measured. Use a trim-pot tool provided in the Accessories Kit to adjust the 15-turn potentiometers for GAIN & SHIFT. The extreme position, indicated by a click, may be located by rotation of at least 15 turns clockwise or anti-clockwise. A two point pressure transducer calibration method can be made as follows: o Connect amplifiers to pressure transducers and switch amplifier ON. o Use a syringe to fill the pressure transducers with distilled water. o Adjust the SHIFT control to give -6.0 volts output at 0 mmhg gage pressure (atmospheric). o Apply a pressure of +200 mmhg to transducers and adjust GAIN for +6.0 volts output. o Repeat steps 4 and 5 to achieve the stated outputs. o CALIBRATION = voltage/pressure = 12 V/200 mmhg = 0.06 V/mmHg. NOTE: The GAIN and SHIFT settings may need to be changed depending on the range of pressure generated in the specific application. Different settings may be needed for monitoring the ViVitro Labs Inc. Pulse Duplicator System. Changing GAIN will also change the output at atmospheric pressure. It may be necessary to re-adjust SHIFT until the required outputs are obtained at the two calibration pressures. Document No V1.3 ViVitro Labs Inc Page 56 of 118

57 9. ViViGen Software 9.1 Installation Install the ViVigen Installer.exe on the computer. This executable program can be found on the supplied flash drive. During installation setup, check the box Create a desktop icon. 9.2 Method Connect the SuperPump Amplifier to the computer using the USB cable. Turn on the amplifier. Open the ViViGen shortcut icon on the desktop. Click the label on the upper left corner to access the drop-down menu. Select Connect... and select a COMX value. If the connection is successful, the red dot will turn green. Click the label on the upper left corner to access the drop-down menu. Document No V1.3 ViVitro Labs Inc Page 57 of 118

58 Select Refresh Waveforms to upload current waveforms from the amplifier to the Waveform Generator console. The amplifier and the console are able to access five waveforms. 9.3 Icons The left side of the console panel shows five index positions. These correspond to the five waveforms stored in the amplifier. It is possible to modify these waveforms using the four icons and the drop-down boxes for each position. Use the Open File icon to search for a stored waveform in the software package. Open the file and view the waveform on the graph. Use the Tools icon to modify or edit the waveform, its label or its Beat Rate. Be sure to define the Type and Duty Cycle, under the Edit Waveform Tools. Note: The Beat Rate has a max value of 400. Do not use a beat rate higher than 220 when running the SuperPump. Use the Save icon to save the modified waveform to a file. Use the Refresh icon to view your changes. Note: Remember to save your waveform, before using the Refresh icon, when making changes under Edit Waveform Tools. Use the Send icon to send the waveform to the amplifier. Note: Do not attempt to Refresh or Upload a waveform when the SuperPump is running. 9.4 Status Request Use the Refresh icon beside the SuperPump Status label to update the screen view with the current status of the pump. Note: the amplitude number represents a percentage value, not a stroke volume. Waveform index gives the index number of the waveform currently being used by the amplifier. Sync pulse delay is given in milliseconds, and can be adjusted using the amplifier controls. Observe the error code box, under the SuperPump Status. Any communications failures or other problems will be signalled here. Note that an attempt to communicate with the amplifier will be repeated until successful or the software is terminated. Note: If the USB is disconnected and then reconnected, the Refresh Status will not update. The SuperPump must be reconnected using drop-down menu, in upper left corner. Document No V1.3 ViVitro Labs Inc Page 58 of 118

59 9.5 New Waveform Generation New waveforms may be created using the drop-down menu from the upper left corner. label in the Select New and complete the requested data in the Create New Waveforms window. Note that 1024 is the standard and expected number of Points. Enter its Label name, its Beat Rate, and its Waveform Type. Select the Save icon and name the file appropriately. Select Done when completed. The new waveform may now be accessed from one of the five index positions, and the duty cycle and beat rate adjusted as required. Document No V1.3 ViVitro Labs Inc Page 59 of 118

60 10. ViViTest Software Operation Pulse Duplicator System User Manual 1. Set up the Pulse Duplicator System (refer to previous sections). 2. Double click on the ViViTest icon located on your desktop. 3. Select the preferred calibration file. (Calibration.cfg is the ViVitro Labs default file with default values to run the software.) NOTE: Good practice indicates that to verify the configuration of the system users should test a valve with known characteristics at the start of every session. Once users are familiar with the waveform image associated with a standard valve, inconsistent waveforms from test valves can be quickly recognized even before analyzing numeric results. The.ACQ data file associated with the standard valve can be loaded in either the Analysis or Review pages in the ViViTest application. In most standard test situations waveform pressure ranges should correspond to physiologic pressure ranges Calibration Process Overview NOTE: It is advised that calibration is carried out each time the software is restarted and at key intervals during use. NOTE: First time through, perform Pump calibration FIRST. Users can select any of 4 types of calibration to perform: Measure sensor zero references Calibrate the flow meter and flow probe(s) (Mitral/Aortic) Calibrate and zero the 3 transducer pressure sensors (atrial, ventricular and aortic) Measure pump linear displacement A complete calibration involves going through each of these Calibration Wizards which is outlined in the following sections. NOTE: The calibration values are read from the selected calibration file and are represented within the Session Information Panel and the Calibration Wizard. These values can be altered to reflect the desired values and saved to a new calibration file via the Calibration Wizard. Document No V1.3 ViVitro Labs Inc Page 60 of 118

61 10.2 Calibration Wizard Navigation The Calibration Process is a step-by-step process guided by the Calibration Wizard. The wizard explains each step of the process leading to measured offsets or gains that constitute the calibration values shown in the Sensor Calibrations panel on the right hand side of the screen. Each of the calibration processes has a navigation panel where users have the opportunity to proceed to the next step or accept the values as presented. Should previous steps need to be revised the user may repeat the process or alternately take a step back to revisit the data entered. Close At any time during the calibration process users can abort by selecting the Close button. This immediately cancels the calibration sequence without saving any of the newly input values. Collapse The Collapse button enables the user to momentarily stall the calibration process without losing the values entered. It causes the Wizard to reduce to a thumbnail in the left hand corner of the screen. Wizard Thumbnail Once the Collapse button is selected a thumbnail will appear in the bottom left corner of the screen. Selecting this thumbnail returns the Wizard to its full size once again. Document No V1.3 ViVitro Labs Inc Page 61 of 118

62 10.3 Pump Calibration The pump piston linear displacement is measured for each unit. The factor in mv/mm is provided by ViVitro manufacturing. The value as shown below is used by the ViViTest software to calibrate the pump. The Linear Transducer (Ch 6) value should be pre-set from ViVitro between 195 and 199, follow the ViViTest pump calibration procedure in the Appendix to ensure the correct pump calibration Factor is entered. The Piston Area should match the area listed on the decal on the rear of the SuperPump. Document No V1.3 ViVitro Labs Inc Page 62 of 118

63 10.4 Flow Meter Calibrating the Flow Meter With this calibration, the flow rate through the transducer will be made directly proportional to the piston displacement signal differentiated with respect to time (dl/dt). The latter signal is an output from the pump amplifier. The PROBE FACTOR should be set to the manufacturer s recommendation. See Carolina Medical label. The probe factor, shown in the red circle, translates to a setting of 4.38 on the flow meter potentiometer. Reference the probe factor calibration label provided with the flow probe for proper set-up. Following the calibration wizard instructions, the software will automatically adjust to create a calibration factor for the flow meter signal to equal the pump dl/dt. If a significant drift is noticed in the flow meter conversion factor, the flow meter and probe may require calibration. Three zero crossovers for each signal should be seen on the voltage graph on step three of the Wizard within the Process Display area. Where this is not the case an error window will appear to run the user through the process again. A typical Pulse Duplicator System configuration consists of a single flow sensor so dl/dt is the default selection meaning that overall system flow is calculated using the pump s RPM sensor, and that flow at the un-instrumented side of the model will be indirectly derived from both Channels 4 and 5. Document No V1.3 ViVitro Labs Inc Page 63 of 118

64 1. Set Pump Control settings on the right to desired levels (Typically users would use a cycle rate at 70bpm using a sine waveform of 50%). A sine wave is required for accurate flow calibration. 2. Click Flow under calibration stage on left 3. Follow step by step calibration wizard NOTE: The data summary presented at the end of the calibration collection sequence is used to show the adjustment (k-factor) of the flow channels to the dl/dt channel. It is important to determine internal acceptance criteria for the flow channel calibration. The software automatically adjusts the flow channel k-factor to equal the dl/dt channel. Document No V1.3 ViVitro Labs Inc Page 64 of 118

65 NOTE: To calibrate the flow transducer using the ViVitro Labs Pulse Duplicator System place an open spacer ring in the flow probe site and an occluding solid plug located in the opposite site. These items are provided in the Pulse Duplicator Accessories Kit. (There should be no compliance between pump piston and flow probe). ViViTest Open Spacer Ring and Solid Occluding Plug for Flow Calibration In cases where flow transducers are mounted in both the aortic and mitral position, the dl/dt signal can be ignored in favor of direct flow measurement at the valve. Document No V1.3 ViVitro Labs Inc Page 65 of 118

66 10.5 Pressure Calibration Calibrating and Zeroing the Sensors Calibration of the aortic, ventricle and atrial pressure sensors has two parts: a 2 point pressure calibration, then a measurement of static pressure head differences between the 3 pressure transducers. NOTE: 2 point pressure calibration pressures must be applied to all 3 transducers simultaneously. NOTE: The low and high pressures should be chosen to span the expected working range of the transducers, e.g. 0 to 200 mmhg. NOTE: Usually the 3 transducers are mounted on a manifold and first exposed to the atmosphere to establish the lower end of the range. Then a large syringe and a digital manometer are used to apply the upper range pressure. The software measures the ADC voltage at each of these limits and uses these as a 2 point slope in calculating the calibration gain. Document No V1.3 ViVitro Labs Inc Page 66 of 118

67 In the ViVitro Labs Pulse Duplicator System, wall pressure measurement sites are not at the same level. Experimental determination of the static head pressure differences is the second part of the pressure calibration. The Wizard prompts the user to record pressure transducer outputs when they are once again installed in the model: first with atmospheric, reference pressure applied, and then with the static head of fluid pressure applied. Application of these pressures is readily made by turning the T taps on the transducers. For custom systems, where pressure transducers are at the same head level, zero static head pressure offsets can be entered manually. NOTE: The calibration factors presented at the end of the calibration collection sequence are related to the accuracy of the pressure data collection. It is important to determine internal acceptance criteria for the pressure transducer calibration factor. The integrity of your data and quality of the results are related to the degree of acceptable resolution. The data accepted should be reviewed to ensure the calibration specification meets the testing objectives. Document No V1.3 ViVitro Labs Inc Page 67 of 118

68 10.6 Zero Calibration Measuring Sensor Zero References Zero offset measurement is the most common of the calibration processes and best practice is that this be done frequently within a valve trial session. Zero values for the sensors are calculated for the pressure, pump and flow sensors on a quiescent system open to atmosphere. Users initially have the option of entering known values directly into the editable fieldsin the Calibration Wizard or follow instructions given by the Calibration Wizard to measure zero voltage values using ADC input. The software tracks the changes of transducer zeros against previously stored zeros. These are indicated as the Drift value in the Sensor Calibrations panel. NOTE: Abnormal zero DRIFT may indicate a malfunctioning transducer Finishing Calibration Updating the Calibration File After all the calibration processes have been completed select the Finished button to save the calibration parameters regenerated in the wizard. At this point you have the option to print the calibration report. This will be saved in the Files folder as an.htm file. Whenever a new calibration is completed the file is rewritten and a new valid checksum calculated Acquisition Process The Acquisition Process allows operators to capture 10 successive waveforms (cycles) for the valve being tested for subsequent analysis by the Analysis Process. The Process Display area displays pressures, flow, transvalve pressures, and pump stroke volume. The Process Controls modify the pressure display. The Support Information and Data Dashboard areas have a number of data boxes showing critical metrics such as cardiac output and pertinent pressures, volumes and flows. NOTE: During the Live Acquisition Phase, voltages measured by the I/O Module are rendered as 6 pressure and flow traces in the Process Display area every ~2.5 seconds each trace corresponds to an A-D channel. Users can hide unwanted traces by selecting one or more of the selector buttons along the left side. Document No V1.3 ViVitro Labs Inc Page 68 of 118

69 10.9 Standard Valve Verification Adjust the Pulse Duplicator System until the quality of the waveforms shown in the graph panel is considered valid. Complete the fields in the Session Information panel. It is particularly important that users indicate the location of the Flow sensor so that flow algorithms operate properly. NOTE: Trial date is filled in automatically. Click the button to capture 10 consecutive valve pulse cycles Save the captured data to an.acq file. The Data Dashboard area shows the data for both the aortic and mitral parts of the heart cycle. NOTE: Once users select the target valve on the right, the opposite control valve data is deemphasized. Snapshot Re-zero Sensors At any point users can select the Snapshot button in the Top Menu Bar to capture a.jpg image of the graph to record particular features of valve behaviour. At any time during a test, users have the option of returning the system to a quiescent state and collecting sensor zeros. This is helpful in confirming sensor accuracy and drift Analyze Process Once one or more 10-cycle ACQ files have been saved users can begin to perform a more comprehensive analysis of the valve cycle waveforms by selecting the Analyze tab an open file dialog allows users to select a valve trial from the list of ACQ files contained in the C:\Program Files\ViVitro Labs\ViViTest\Files folder on the right. The left side selector buttons allow users to select one, several or all of the 10 acquisition trial waveforms for graphing. NOTE: When the All Waveforms (cycles) button is selected, statistics shown on the graph and in the lower results panels apply to the first waveform by default. To see the results of another waveform, simply click once on the waveform number you wish to see. When some but not all of the waveforms are selected statistics apply to the most recent waveform selected. In all cases data in the results panel is a mean of 10 cycles. The [data table] contains details for each waveform. Data table button located at the top of the screen Document No V1.3 ViVitro Labs Inc Page 69 of 118

70 10.12 Crossovers and Interval Identification Data analysis proceeds sequentially from cycle 1 to 10. Pressure and flow waveforms are displayed along with crossover markers defining critical intervals during the cycle. Automatic algorithms calculate the start and end of the aortic and mitral valve pressure and flow cycles. The Data Dashboard is based on these crossover points. NOTE: While the algorithms make reasonably good choices on well formed waveforms do not blindly rely on this automatic placement. Manually adjust the crossover markers by clicking and dragging on their labels to change their horizontal location to get best results. Adjustable cross over markers Overlaid gradient bars at the top of the graph mark pressure intervals - bars at the bottom of the graph mark flow intervals. NOTE: Markers must be in correct logical sequence from left to right. If this is not the case, analysis will stop and an error bar will appear below the graph. Markers must then be moved to be in sequential order. Document No V1.3 ViVitro Labs Inc Page 70 of 118

71 The end of the Closing Flow interval may need adjustment, particularly if the flow is non-zero at these points. There may be a precursor to positive flow in which case the start of forward flow could be advanced in time to avoid negative values of mean transvalve pressure during the forward flow interval. Users should check the crossover placements for each of the 10 waveforms that makeup the analysis data. NOTE: When All Waveforms is selected, moving markers will affect all 10 waveforms Producing Data Output Once analysis and adjustment of 10 cycles of data has been completed, means and standard deviations for the 10 cycles can be output as an.ana file,.csv file, data table or printed report by selecting the Save, Export CSV, Data Table or Print icons (see the Appendices for further instruction). As in the Acquisition Process, graph traces may be hidden by selecting their trace button below the graph. Users can define the graph s level of complexity by selecting trace names in the Pressures and Flows Legend to highlight or hide traces Derived Flow Measurements Total System Flow Flow Meter Flow = Un-instrumented Valve Flow This derived flow will only be meaningful if the data was acquired using the ViVitro Labs Pulse Duplicator System with no compliance between pump piston and the model s ventricle. In this case, the derived flow should correspond to the flow in the side without a flow probe, either aortic or mitral Useful Icons Save File Refresh Use the refresh button in the upper right corner of the graph to clear any artifacts from the graph should it become corrupted. To change pressure units for the values shown in the Data Dashboard, select the mmhg / KPa toggle at the upper right corner. Static head pressure compensation is one of the parameters measured during the sensor calibration process. Users can enable or disable these 3 pressure sensor compensation values to the data algorithms by selecting the Static Head compensation checkbox in the Sensor Calibrations panel. Once satisfied with the results, save the Analysis means for the 10 waveforms to an.ana file by selecting the Save button. Data Table Users can review the results calculated for each of the 10 waveforms in detail by reviewing a data table showing all 10 waveforms and the cumulative means. Select the Data Table button in the title bar to bring up the data table. Document No V1.3 ViVitro Labs Inc Page 71 of 118

72 Export CSV Printed Report To put the same calculated results into an editable file for later off-line analysis select the Export CSV icon this creates a comma separated value (CSV) file. Users can create a report of the Analysis by selecting the Print icon to write an HTML file to the Files folder. Verify the data shown in the on-screen report review. NOTE: Files generated by the software such as CSV files or HTML report files are written to C:\Program Files\ViVitro Labs\ViViTest\Files unless otherwise directed Compare Process The Compare Process allows users to compare analysis for a series of valve trials: for one valve against a similar series of trials for a second, third, valve. Features of the Compare Process interface: Main Process Control select categories of mean data to be plotted, Various Y axis data types can be selected by selecting the direction buttons flanking the Y axis label, Various X axis flow data types can be selected by selecting the direction buttons flanking the X axis label, The range of the X and Y axes are controlled by the zoom buttons at the right or top end of these axes, The Data Dashboard is split between a pair of trials selected from 2 groups. The following data types may be shown: Area - Graph displays curve of selected groups for Area (P - pressure), (PF pressure and flow), (F - flow) Energy - Graph displays curve of selected groups for Energy (Forward), (Closing), (Closed), (Total), in mj. Pressure - Graph displays curve of selected groups for Pressure (Mean Back), (Peak Differential), (Mean Positive), (Mean Differential forward), (Mean Differential hybrid). Document No V1.3 ViVitro Labs Inc Page 72 of 118

73 Regurge - Graph displays curve of selected groups for Regurgitation (Total), (Percent), (Closing), (Closed), and (Leakage Rate). NOTE: There is no Legend bar for this interface. See the help screen for more information Comparing Groups of Trials Groups are created by selecting the Groups of Trials buttons in the top of the Analysis Groups panel at screen right. The sequence of creating groups for comparison is as follows: select either aortic or mitral Valve Data, select the checkbox to of each.ana file required to create a group with a default name or edit the Create Group box for a custom name, add ANA files to the group by selecting the checkbox next to the file name in the Analysis Files list, repeat the steps above to create another group, NOTE: If the groups tree need to be cleared select the Clear button. Save the group using the save button and enter a file name in the Save dialog box. The new groups file appears as a.grp name in the Analysis files list Auto-scaling and Zooming When users select a different Y graph type or change the data type selection shown in the X or Y axis the graph is auto-scaled to fit the data before it is rendered. Users can override and adjust this auto-scaling using the zoom buttons for either axis Handling Compare Results Export CSV Print Report To put the list of trial means being compared into an editable file for off-line analysis select the Export CSV button this creates a comma separated value (CSV) file. Users can create a report of the Compare means by selecting the printer icon in the title bar to write an HTML file to the Files folder. Verify the data in the report review and either delete the HTML file or print to the system printer from a browser. Document No V1.3 ViVitro Labs Inc Page 73 of 118

74 10.20 Review Process The Review Process allows users to browse acquisition (.ACQ) files and quickly graph their pressure, flow and differential transvalve pressure and power characteristics on a common timescale. The list of current.acq files is shown in a checked list in the Selected Files panel at screen right I/O Module Re-initialization In cases where the I/O Module needs re-initialization the following sequence is recommended: 1. Remove the I/O Module USB cable from the PC. 2. Wait 10 seconds and then re-insert the USB cable to the PC. 3. Navigate to InstaCal icon on your desktop and double click 4. Highlight the board number in the detection box and click OK 5. Right click on the board number in the InstaCal box and select configure. 6. Confirm settings are set to: a. 8 differential channels b. Factory calibration c. XAPCR Edge = Rising d. XAPCR Pin direction = input e. XDPCR Edge = Rising f. XDPCR pin direction = input g. ADC settling time = 10us 7. Click OK 8. Right click on the board number in the InstaCal box and select Calibrate A/D 9. Click Calibrate 10. Click OK and Exit program Running a Report ViViTest reports capture a moment in time during a valve s trial. Results displayed on the report enable the user to identify the trial, valve and selected value used for the trial. A screenshot of the Process Display area is provided for visualization of the waveforms. Using the Print icon a report is created within an HTML document. These files are automatically saved to your Files folder within the software directory. NOTE: The report will open immediately to screen. A copy will have automatically been saved to the Files folder. To Open and Print: To keep a physical copy of the report users can: 1. Access the file via Explorer under C:\Program_Files\ViVitro_Labs\ViViTest\Files. NOTE: The file will be identified with.html format. The file name will be the identifier information entered for the valve and the date and time. Open the desired report. Document No V1.3 ViVitro Labs Inc Page 74 of 118

75 NOTE: This will open in a Browser or the computers preferred.html file viewing program. Select to print from the menu Taking a Snapshot Snapshots enable the user to display represented graphs of the trial s valve cycle activity waveforms at any moment. Click the Snapshot icon to produce a.jpg file of the Process Display area. Extra data is included in the file in the top right corner of the graph to identify trial, valve and other related data. Customizing the Snapshot: The Process Display area can be manipulated to zoom in via toggling of the tool options within the graph. To Save, Open and Print: 1. Selecting the Snapshot button will capture the Process Display area as it is seen on the screen. A save window will display to enable the user to customize the file name, alternately the user can opt to use the automatically assigned file name created from valve identifier information, date and time. NOTE: The Snapshot will not open immediately but be saved to the Files directory of the software for later viewing. 2. Access the file via Explorer under C:\Program_Files\ViVitro_Labs\ViViTest\Files. NOTE: The file will be identified with.jpg format. The file name will be the identifier information entered for the valve and the date and time or the customized name the operator assigned. 3. Open the desired Snapshot. 4. Select print from the menu Exporting CSV file data For researchers/developers wishing to extract the data to create algorithms and calculations to provide specific results the option of producing a raw data.csv file is available. Users can access this under the Analyze tab either as raw data or by opening a results table and exporting the calculated data. NOTE: The output presents as many cycles as displayed within the Process Display area. By clicking on the Export CSV icon you can create a.csv file once data has been accumulated. The output is suitable to be viewed and manipulated within a.csv file reading program. Data supplied includes trial and valve information as well as: Associated data creating the current trial scene; Raw collected Inflow/Outflow pressure measurements throughout the displayed cycles. To Save, Open and Print: Document No V1.3 ViVitro Labs Inc Page 75 of 118

76 1. Selecting the Export CSV button will capture the data associated with the Process Display area as it is seen on the screen. A save window will display to enable the user to customize the file name, alternately the user can opt to use the automatically assigned file name created from valve identifier information, data and time. NOTE: The.CSV file will not open immediately but be saved to the Files directory of the software for later viewing. 2. Access the file via Explorer under C:\Program_Files\ViVitro_Labs\ViViTest\Files. NOTE: The file will be identified with.csv format. The file name will be the identifier information entered for the valve and the date and time or the customized name the operator assigned. 3. Open the desired.csv. NOTE: This will open within the computers preferred.csv file viewing program. 4. Select print from the menu ANA file During the Analyze Process an.ana file is created to store the save data collected. The.ANA file enables the operator to open the analysis information to allow comparisons. To Save, Open and Print: 1. Using the Save button save the current data to an.ana file. A save window will display to enable the user to customize the file name, alternately the user can opt to use the automatically assigned file name created from valve identifier information, data and time. To retrieve the.ana file select the desired analysis files on the bottom right side of the software under the directory C:\Program_Files\ViVitro_Labs\ViViTest\Files. NOTE: This action will enable the operator to carry out print, snapshot and excel export activities pertaining to the data saved Log File As an ongoing background feature of ViViTest, log files are created each time a user instructs the software to do an activity. These files can be accessed via the C:\Program_Files\ViVitro_Labs\ViViTest\Logs directory as a.log file. To Open and Print: 1. Access the file via Explorer under C:\Program_Files\ViVitro_Labs\ViViTest\Logs. NOTE: The file will be identified with a.log format. The file name will be the identifier information entered for the valve and the date and time. 2. Open the desired.log. NOTE: This will open within the computers preferred.log file viewing program. 3. Select print from the menu Waveform Design Users can change waveform designs by clicking on and selecting Edit Pump Waveforms. Designing Custom Waveforms: Document No V1.3 ViVitro Labs Inc Page 76 of 118

77 Waveforms Users with special requirements may be interested in designing new custom waveforms. These 4 waveform buttons allow users to load and modify a dual-sine waveform, a 2-line waveform, a spline waveform and a wavetrain. After a custom waveform is designed it is then saved as a.wvf or.wvt file. Users create a custom sine or 2-line waveform by dragging the bottom point of the 2-line V or dual sine valley in the X direction until the desired shape is achieved. ViViTest Waveform customization custom sine wave Spline waveforms are created by dragging one or more of the spline points to create a new shape. New spline points are created by clicking on the line whereas points can be deleted by right clicking on the point. Users select a point along a segment and then drag that point up or down in the Y direction, until the desired shape is achieved. Document No V1.3 ViVitro Labs Inc Page 77 of 118

78 ViViTest Waveform customization spline wave Wavetrains are created by selecting one of the saved waveform from the waveform design files on the right hand side. Once the file has been selected, click on the first box (or desired location) in the Wavetrain Design window below the graph. Users can create a train consisting of a variety of waveforms up to 10 in sequence. (i.e. location 1-S30.WVF, location 2-S35.WVF, location 3-S40.WVF.) ViViTest WaveTrain Document No V1.3 ViVitro Labs Inc Page 78 of 118

79 11. Software Interface Orientation Pulse Duplicator System User Manual ViViTest Screen Layout 11.1 Top Level Toolbar Standard controls in the top level toolbar are: Open File Open an Acquisition (.ACQ) file for analysis or Analysis (.ANA) file for comparison graphing. Save File Snapshot Print Export CSV Data Table Used to save an Acquisition file when in Acquisition Process. Captures a (.JPG) screen snapshot of the user interface. (See Tooltip for details) In Analysis or Compare processes users can produce a report (.HTML) of results which can then be viewed and printed in any browser. Save Acquisition or Compare data to a comma separated value (CSV) file for further processing. In Analysis process the 10 sequential cycles saved to an.acq file are analyzed and the resulting means data shown in tabular format. Document No V1.3 ViVitro Labs Inc Page 79 of 118

80 Options Menu Minimize Exit Access to all software features and information about ViViTest. Minimizes the software to the windows toolbar to enable the user to carry out other activities on the computer screen. Closes the application. NOTE: Most controls on the user interface have tooltips that give a brief explanation of usage whenever the mouse is hovered over the control. NOTE: All files saved from the software are saved to the Files folder of the software directory. (e.g. C:\Program Files\ViVitro Labs\ViViTest\Files) 11.2 Process Selection and Secondary Buttons These tabs transfer you to the various components of the software. These are: Calibration Process, Acquisition Process, Analysis Process, Compare Process and Acquisition Review Support Information Area Demographic statistics, process table data and context help in collapsible panels at screen right. Displays editable and non-editable information relating to the Process Display area. Collapses the selected data area. Opens the selected data area. Session Information Set up key valve identifier information for each time a new valve is being tested. Ensure that the correct flow probe site is selected to gain correct readings. Data entered within this area will be used to create automated file names. Sensor Calibrations Represents the collected calibration values within the selected calibration file. Enable/disable Static Head compensation to alter readings as desired. Document No V1.3 ViVitro Labs Inc Page 80 of 118

81 Acquisition Files Allows access to all.acq files saved for previous valve trials. Analog Voltage Information Provides analog voltage information for the flow probe and transducers used within the system. Pressure Calibration Values As pressure calibration is carried out the values are represented within this area for reference. Flow Calibration Values Provides the flow probe calibration values for reference. Analysis Groups Allows the user to create groups of valve trial data for analysis and comparison. Users have the ability to select.ana files from the list below to assign them to a group. Groups can be cleared and created easily within this area and are saved as.grp files. Zeroing Calibration Values Provides pressures, flow and pump zeroing calibration values for reference. Document No V1.3 ViVitro Labs Inc Page 81 of 118

82 Pump Displacement Calibration Displays pump linear transducer calibration values as input in the Wizard and represented within the.cfg file Main Process Controls Control buttons which modify or add attributes to the Process Display area. Calibrate Acquire Analyze Compare Review 11.5 Data Dashboard Information relating to Aortic or Mitral Valve measurements. Provides non-editable summary data for both the aortic and mitral valves, Measurement units can be toggled by using the mmhg / KPa button. Document No V1.3 ViVitro Labs Inc Page 82 of 118

83 11.6 Process Display Displays the activity of information gathered. In most cases the following will be displayed: a multi-axis graph typically showing both pressure and flow data over a 1.35 pump cycle most recent pressure sensor values for most recent ADC analysis multiple valve cycles A legend bar indicating the meaning of the various coloured graph traces. These can be turned on/off to highlight specific data to be viewed: o RED line Aortic Pressure o YELLOW line Ventricular Pressure o BLUE line Atrial Pressure o PINK line Transaortic Pressure o ORANGE line Transmitral Pressure o WHITE line Derived Flow o GREEN line Pump dl/dt o DARK BLUE line Flow meter o GREY line Pump Volume The graph can be manipulated by: Refresh this allows the data to be refreshed/reloaded to display the latest data. Restore enables the changes done to the graph view to revert to 1:1. Increase/Decrease (zoom) manipulates the interval frequency of the measured pressure to bring the graph closer or further away. Up/Down enables the user to position the adapted graph data more centrally on the screen by adjusting the Y axis. Document No V1.3 ViVitro Labs Inc Page 83 of 118

84 11.7 Process Output The software uses a number of various calculations to output accurate and relevant information for your review. Some of the standard formulas have been listed below for your ease of reference. Please reference Appendix E for a more complete list of calculations used. For anything further please contact ViVitro Labs directly. Q rms p 51.6 Q rms t 2 t1 Q( t) t 2 t 2 1 dt 11.8 Help Information pertaining to the screen or the software in general Help is available throughout the use of ViViTest. Document No V1.3 ViVitro Labs Inc Page 84 of 118

85 12. System Maintenance 12.1 Required Fluid There are 3 zones of your system that will require differing fluid requirements. 1. The main operating zone: within the Model Left Heart, Peripheral Resistance Controller and connecting tubes. 2. The pulse duplicating zone: within the pump head, VIA (if installed) and Ventricle Chamber. 3. The heat exchanging zone: within the heat exchanger, heat bath, heat pump and tubing. Zone 1 Depending on your requirements the following fluid information is provided as a guide for use in the system. Fluid: Saline; or A blood analogue fluid consisting of ~35% glycerin 65% saline by volume. This mixture having a density of approximately g/ml at 20 C will have a viscosity of approximately 3.2 mpa s at 37 C. This approximates the viscosity of blood. Any solution used must be mixed to 0.9% NaCl concentration to operate the electromagnetic flow probes properly. Volume: Approximately 2 liters Zone 2 Fluid: Distilled water mixed with an appropriate biocide may be required to reduce the bioburden impact of contamination. Contact a supplier for recommendations. Zone 3 Fluid: Water mixed with an algaecide to prevent algae growth. NOTE: The fluid surrounding the ventricle in the hydraulic chamber and the VIA fluid is normally distilled water and can remain in situ for a period of time. It should however be replaced when bio-film contamination compromises visibility. In general, replace the hydraulic fluid every 2 months. If the system is not used for extended periods, the hydraulic chamber fluid should be drained and all components cleaned with a soft cotton cloth and detergent and then dried before storage. Document No V1.3 ViVitro Labs Inc Page 85 of 118

86 12.2 Draining Fluid NOTE: Prior to draining the system, ensure the flow meter is turned off. Operatinig the flow meter without fluid in the system will damage the flow probe. To change valves, or to disassemble the system, the test fluid must first be drained. To do this, release the drain tube clamp and collect fluid in a container of ~2 liters volume. Re-clamp drain after system is drained. Fluid remaining in the left atrium reservoir can be drained by temporarily disconnecting the green colour coded hose connected to the left atrium port. Remaining test fluid in the aortic root above the aortic valve can be removed by syringe through a pressure port stopcock. Replacing frequency: Zone 1: Will be replaced by natural course due to the changing of prosthetic structures on a regular basis. Zone 2: Replace every 2 months. Zone 3: Replace every 2 months. Instructions Zone 1: 1. Ensure the system is turned off, including the flow probe/s 2. Place bucket under Ventricle Chamber. 3. Open drain valve situated beneath the chamber. 4. Allow to completely empty. NOTE: Ideally you would allow it to completely dry before placing further fluids in the system. Instructions Zone 2: 1. Ensure the system is turned off, including the flow probe/s 2. Place bucket under Ventricle Chamber 3. Open luer drain port on bottom of Ventricle Chamber. 4. Tip the entire system to 30º with the motor at the higher end. (This will aid the draining of the VIA and pump head). 5. Return to horizontal position. NOTE: Ideally you would allow it to completely dry before placing further fluids in the system. Instructions Zone 3: Ensure the system is turned off, including the flow probe/s. Drain as per the Heat Bath manufacturers recommendations Cleaning Acrylic Fluid The other acrylic plastic parts can be cleaned using a soft cotton cloth with detergent and water. If required, acrylic parts can also be sterilized by using glutaraldehyde or a dilute hydrogen peroxide solution. Keep these solutions away from the flow probe electrodes. Alcohol, abrasives and solvents should never be allowed to come into contact with the acrylic components of the Model Heart. Document No V1.3 ViVitro Labs Inc Page 86 of 118

87 12.4 Aorta Replacement CAUTION: THE GLASS AORTA IS FRAGILE - HANDLE WITH CARE - DO NOT APPLY UNDUE STRESS 1. Remove the original aorta/exit tube assembly from the load assembly: o remove two 2-56 bolts on the clamp retaining the catheter to the side of the aorta exit tube; o remove four 6-32 x 3/8 inch bolts to detach the white plastic plate at the entrance of the glass aorta; o loosen the two 6-32 bolts and rotate the washers near the exit end of the glass o aorta to allow aorta assembly removal; push the exit end of the plastic aorta exit tube to remove glass aorta and exit tube assembly. 2. Remove remnants of the original aorta and silicone sealant from the exit tube. 3. Attach three 2 cm strips of adhesive tape equally spaced on the inside surface of the new aorta exit with 1 cm of tape projecting above the end. 4. Hold the aorta exit tube above the glass aorta so that the pressure port is aligned with a sinus edge. 5. Mate and center the exit tube counter bore with the aorta and then use the tape to hold the two parts together. 6. Use a syringe filled with silicone sealant with attached dispensing tip (e.g. EFD Inc. #5118PPS-B) to fill the gap between the glass and aorta exit tube taking care not to entrap air bubbles which could cause leaks. Any excess sealant should be wiped before curing occurs. 7. Let sealant cure (24 hours). 8. Remove adhesive tape from inside surfaces of assembly. 9. Re-insert assembly into housing reversing the 4 bulleted steps shown above in step Ventricle Member Replacement The model ventricle and bonded drain tube are made of silicone rubber and should have a long flex life. If damaged, they may be repairable using RTV Silicone rubber. Otherwise, replace as follows: 1. Remove 8 X-head bolts and valve block retaining tabs then remove block 2. Remove polycarbonate base filler ring 3. Move ventricle base aside to gain access to the bonded silicone tube and pull off from polycarbonate tube. Reverse above sequence to install new ventricle. Document No V1.3 ViVitro Labs Inc Page 87 of 118

88 12.6 Piston Seal Replacement 1. Remove the three #6-32 x ¾ screws located on the front of the piston and remove the SuperPump Piston Cap (09533). #6-32 x ¾ SHCS Piston Cap 2. Remove the four ¼ -20 x 4.5 screws on the front of the pump head. ¼ -20 x 4.5 SHCS Short Extrusion End Cap 3. Remove the Short Extrusion (08981) and end cap from the pump assembly. M10 Nut and Lock Washer Document No V1.3 ViVitro Labs Inc Page 88 of 118

89 4. Carefully remove the glass cylinder from the piston. CAUTION: DURING THIS STEP BE CAREFUL WHEN HANDLING THE GLASS CYLINDER AS THE EDGES MAY BE SHARP. 5. Remove the M10 nut and lockwasher from the front of the piston. 6. Remove the SuperPump Piston (09532) from the rod end. Piston Seal 7. Locate the small hole near the outer edge of the piston. Holding the piston firmly, press off the piston seal with a small hex key. Be sure not to scratch or damage the piston sealing surface during this step. Piston Sealing Surface 8. Check the sealing surface of the piston for scratches that are either visible or can be felt with a fingernail. Ensuring the orientation is correct, snap the piston seal onto the piston carefully by hand. 9. Clean any dust and debris from the glass cylinder. Apply two pieces of electrical tape to one edge of the glass cylinder. Document No V1.3 ViVitro Labs Inc Page 89 of 118

90 10. Push the piston into the glass cylinder (SPM042) using the tape as a glide point. Remove the electrical tape from the Glass Cylinder (SPM042). 11. Slide rod end into the SuperPump Piston (09532). Fasten in place with a M10 Lock Washer (09538), Loctite, and the nut from the rod end. Ensure the glass cylinder does not bump against the cylinder rod. M10 Nut and Lock Washer 12. Push the Glass Cylinder (SPM042) onto the pump. Ensure that the piston does not come close to the sharp edge of the glass cylinder. 13. Place the Short Extrusion (08981) onto the pump. The gap in the short extrusion should be in line with the longer flat section of the central stand. Short Extrusion (08981) (Note orientation) 14. Push O-Ring-235 (SPM072) into the groove on the outside face of End Cap (08975). 15. Push O-Ring-335 (10084) into the groove on the inside face of End Cap (08975). Document No V1.3 ViVitro Labs Inc Page 90 of 118

91 SPM072 Long flat section Outside Face Inside Face 16. Fasten the End Cap (08975) onto the pump head with four ¼ -20 x 4.5 SHCS (08980). Take care not to chip the edge of the glass with the aluminum parts. Tighten the fasteners gradually to prevent uneven stress on the glass cylinder. Compress the sides of the Short Extrusion (08981) while tightening so that the sides sit flush with the end cap faces. ¼ -20 x 4.5 SHCS 17. Check for damage on O-Ring (09534) into the recess of the SuperPump Piston Cap (09533). 18. Fasten the SuperPump Piston Cap (09533) to the SuperPump Piston with three #6-32 x ¾ SHCS (HCM079). Loctite is not required. The SuperPump is now ready for installation of the Viscoelastic Impedance Adaptor (VIA) or Model Left Heart. Document No V1.3 ViVitro Labs Inc Page 91 of 118

92 Compliance x 10-6, cm 5 / dyne Pulse Duplicator System User Manual 13. Appendix A Supplemental Component Notes The extra compliance available with Large Volume Compliance bottle is useful in applications where aortic root or LVOT compliance is minimal. This will occur in applications where low compliance conduits are being utilized. This is typical when stentless aortic valves, silicone rubber aortas (PVL applications) and LVOT models are involved in the test work. If desired, the air volume simulating characteristic compliance can be reduced by allowing the fluid level to rise higher in the characteristic compliance chamber and/or by adding water to the supplementary bottle as shown below for the optional large supplementary compliance bottle. The diagram depicts how compliance adjustment is accomplished using water to reduce the air volume. Note on compliance modeling Tissue compliance is simulated in the Model Heart and VIA models by using contained air volumes. These air volumes are adjustable and cover a physiologic range for simulating left ventricle, aortic root and systemic arterial compliance. Compliance is defined as the ratio of volume change to pressure difference as follows: The following graph shows experimental data for contained air volumes versus compliance. Initial static pressure 1 P ranged from 0 to 120 mm Hg. Best fit linear equations are given for the four data lines. It can be seen that in a contained air volume, as initial pressure condition increases, compliance decreases. Air volume max values that were experimentally found appropriate for simulating left ventricle, aortic root and systemic arterial compliance are: Left ventricle -Source compliance air volume = 120 ml; -output compliance air volume = 50 ml Aortic root = 640 ml Systemic arterial = 615 ml These air volume compliances helped provide realistic pressure and flow wave forms under pulsatile flow conditions. Air volumes less than these can simulate various patho-physiologic or stiffened tissue conditions where tissues may exhibit less compliance y = 0.983x R² = Air Volume Compliance (VSI\Air Compliance Calib.xls, 29 Jan., 13 Feb., 2002 TNS/LNS) Initial Pressure P1 (mm Hg): y = 0.913x R² = y = 0.880x R² = y = 0.875x R² = Air Volumemax, ml Document No V1.3 ViVitro Labs Inc Page 92 of 118

93 14. Appendix B Installing LVOT Accessories (Optional) The ViVitro Labs Inc. Pulse Duplicator System and aortic system has been used by some clients to test stentless aortic valves and simulate left ventricular outflow tracts (LVOT). Although compliant aortic roots with sinuses are not currently provided by ViVitro Labs Inc., this instruction describes a method whereby optional ViVitro Labs Inc. flanges may assist in mounting the researcher s own structures into the ViVitro Labs Inc. Pulse Duplicator System. NOTE: The diameter of a compliant aorta will change under dynamic flow and pressure conditions. The minimum diameter of the aorta during pulsatile flow testing should be approximately that of the relaxed or molded diameter of the aorta. This can be achieved by adjusting the mean pressure on the outside of the aorta using a syringe to inject or withdraw air from the aortic root compliance chamber. A10 A8 A66 A67 FIGURE LEGEND: A8 Catheter clamp (with V-006 O-ring) & 2-56 x 3/8 inch s.s. bolts, 2 ea. A10 High pressure catheter tube A35 Aorta clamp washers & 6-32 x 3/8 inch s.s. bolts, 2 ea. A62 A62 Silicone O-ring (shown bonded to compliant aorta exit): A63 Clamping bolt (for part #8), 6-32x¾ inch, 1 each A63 A69 A68 A64 Compliant silicone aorta/rvot no sinuses (13-28 mm ID) A64.1 Stentless Aortic valve mounted to aorta (user supplied) A65 Silicone rubber inlet ring (bonded to silicone aorta/rvot) A66 Outlet tube (male) for Flex aorta: A67 Clamping bolts 6-32 x ¾ inch, 3 each A64 A35 A68 Outlet tube clamp for Flex aorta, (common for all sizes) A69 Outlet tube (female) for Flex aorta: A70 Bond between aorta and aortic inlet ring A70 A64.1 A65 NOTE: It is recommended that these structures be made from silicone rubber. A silicone rubber aorta will bond easily to the other silicone rubber components that are needed using a silicone rubber sealant. 1. Custom make the aortic inlet ring from silicone rubber following procedures given in this manual. 2. The aortic ring is then bonded to the inflow of the aorta using Dow Corning flowable silicone sealant #734. NOTE: A silicone rubber O-ring is supplied and is bonded to the outlet of the compliant aorta with the same sealant. NOTE: The grey and green tubes are supplied in various ID sizes with internal diameters (13-28mm) NOTE: Appropriate sized silicone rubber O-rings are also supplied for bonding to the exit end of the aorta. 3. Before mounting the compliant aorta/stentless valve and accessories, remove the standard glass aorta assembly following the instruction in the operating manual for aorta replacement. Document No V1.3 ViVitro Labs Inc Page 93 of 118

94 4. Bond a silicone O-ring {A62} to the exit end of the desired compliant aorta and mould a silicone rubber inlet ring {A65} with an inside diameter matching the inflow end of this aorta {A64}. 5. Select matched sized tubes {A66} and {A69} appropriate to the size of the aorta being attached. 6. With the clamp {A68} detached, insert the green tube {A69} into the aortic assembly housing and secure with the two clamping washers and bolts {A35}. 7. Place the outlet tube clamp {A68} over the green tube {A69}, temporarily leaving the bolt {A63} loose. 8. Attach the catheter {A10} to the grey outlet tube {A66} using the catheter clamp and bolts {A8}. 9. Mate the grey {A66} and green {A69} tubes and rotate the yellow clamp {A68} so that the three bolt {A67} holes align. 10. Tighten the clamp screw {A63}. 11. Insert the aorta exit O-ring {A62} between the grey {A66} and green {A69} tubes and clamp using the three supplied bolts. 12. Insert the inlet of the aorta into the silicone rubber inlet ring {A65} and before bonding the two components together, assure that there is no twisting or buckling of the aortic walls. 13. Once the bond is secure, remove the aorta from the assembly and proceed with mounting the stentless aortic valve into the compliant aorta. NOTE: This process may involve suturing the valve the inside of the aorta. Resulting suture holes and sutures can be reinforced and sealed with a bead of silicone sealant on the inside and outside of the aorta. In this regard, silicone rubber sealant will vulcanize in water and therefore allows tissue type stentless valves to be kept moist. 14. With stentless valve and aorta assembly complete, install assembly into the Pulse Duplicator System aortic assembly housing and proceed with function testing Document No V1.3 ViVitro Labs Inc Page 94 of 118

95 15. Appendix C LVOT Simulation Assembly (Optional) This sketch depicts a mounting scheme using silicone tubing to simulate a variety of LVOT sizes. Parts A66, A68 and A69 are custom fitted to accommodate silicone tubes with relaxed tube ID s of 13, 16, 19, 22, 25, and 28 mm. The 28 mm tube may be expandable up to about 50 mm OD and cover intermediate sizes as indicated MAXIMUM INTERNAL DIAMETER = 28 mm 79.4 RVOT outflow pressure port 12.7 A66 A dia. 7.0 A69 ~ SCALE: ~10 mm RVOT inflow pressure port dia. USING A SYRINGE FROM THIS PORT, SUCTION REMOVAL OF AIR or WATER SURROUNDING THE SILICONE TUBE RVOT MODEL ALLOWS DESIRED DIAMETER TO BE OBTAINED. USING THE UNEXPANDED 28mm TUBE SIZE, ALL INTERMEDIATE SIZES (28-50 mm Dia) COULD LIKELY BE OBTAINED. VIVITRO IncSYSTEMS Victoria, B.C. Canada Ph: Fax: SCALE: X1 APPROVED BY: DATE: 7 FEB 2007 L.N. SCOTTEN DRAWN BY: REVISED: 16 OCT 07 SKETCH OF POTENTIAL EXPANDABLE RVOT OUTFLOW TRACT FOR NECKER, HOSPITAL, PARIS MATERIALS: VARIOUS ALL DIMENSIONS ARE GIVEN IN MILLIMETRES UNLESS OTHERWISE STATED DRAWING NUMBER: SDM070207A Document No V1.3 ViVitro Labs Inc Page 95 of 118

96 16. Appendix D Components and Parts Lists 16.1 SuperPump 1 SuperPump (includes pump head and motor pre-installed) 1 SuperPump Motor power supply cable 1 SuperPump Amplifier 1 SuperPump Amplifier power supply cable 1 Cooling fan 1 SuperPump mounting shelf Pulse Duplicator System User Manual 16.2 Model Left Heart 1 Ventricle chamber 1 Aortic chamber 1 Atrium chamber 1 Aortic standoff 1 Accessories kit (includes 2 compliance chambers, 1 peripheral resistance controller and 4 colour coded silicone tubes) 1 Accessories table 2 Mechanical disc valve 16.3 Data Acquisition System 1 I/O Module 1 Computer and associated components 1 ViViTest Software package 1 USB cable 3 Short BNC cables 3 Long BNC cables 16.4 Flow Measuring 1 Flow Measuring controller 1 Flow probe 1 Flow probe cable kit (includes cable to flow probe and ground) 16.5 Optional System Components Heat Exchanger 1 Heat bath including pump and controller box 1 Heat Exchanger 2 Long silicone tubes 1 Short silicone tube 1 Power supply cable Manometer Document No V1.3 ViVitro Labs Inc Page 96 of 118

97 17. Appendix E Glossary Test Fluid Fluid used within the chambers, pump and pump head. Aortic Chamber Provides (in conjunction with the Aortic Root Compliance Chamber and Aortic Compliance chamber) characteristic resistance which simulates the left aortic functions of the heart. Atrium Chamber Provides characteristic resistance which simulates the left atrium functions of the heart. Aortic Root Provides (in conjunction with the Aortic Chamber and Aortic Compliance Compliance Chamber Chamber) characteristic resistance which simulates the left aortic functions of the heart. Aortic Compliance Chamber Provides (in conjunction with the Aortic Chamber and Aortic Root Compliance Chamber) characteristic resistance which simulates the left aortic functions of the heart. Mean Atrial Measurement derived from the pressure created between the Atrium Pressure Chamber and the Ventricle Chamber to simulate the left atrial pressure in the heart in the mitral heart valve region. Measurement is gained using a Mitral Flow Probe. Aortic Pressure Measurement derived from the pressure created between the Ventricle Chamber and the Aortic Standoff to simulate the left aortic pressure in the heart in the aortic heart valve region. Measurement is gained using an Aortic Flow Probe. Ventricular Pressure created in the Ventricle Chamber prior to the Aortic heart valve Pressure DL/DT opening. A signal provided by the SuperPump Amplifier indicating the pump s flow rate in liters per unit time. dl/dt pump flow data represents overall system flow and can be used in conjunction with the flow meter to derive the flow across the valve opposite to the flow meter. Document No V1.3 ViVitro Labs Inc Page 97 of 118

98 18. Appendix F Pump Calibration Procedure 18.1 Resetting Values 1. Run ViVitest Software. Select the Calibration Tab. Open the calibration file: Calibration.cfg. 2. Turn the amplitude to zero. 3. Select the Pump calibration tab. 4. Change the Linear Transducer (Ch 6): value to mv/mm. Verify that the Piston Area is set to cm2. Select Accept. 5. Select the Calibrate Tab. This saves changes to the calibration file Calibration.cfg. Close the print preview dialog box. Select the Calibrate Tab. Open the Calibration.cfg file. (this process saves the mv/mm value). Document No V1.3 ViVitro Labs Inc Page 98 of 118

99 18.2 Determing Interal Pump Calibration Value 1. Turn the SuperPump Actuator on. Set the internal waveform to S50_70. Adjust the amplitude knob to an arbitrary value and watch to ensure ViViTest is showing a sine waveform for the Pump Vol signal. All other signals can be hidden if desired. 2. Turn the SuperPump amplitude knob up the values described in the table below. Record the controller stroke volume for both the controller display and the ViViTest display. Calculate the values for the right hand column. Average the results of the 3 calculations. (should be ~ 197 ml/stroke) Controller Stroke (ml/s) ViViTest Stroke (ml/s) = (ViViTest value / Controller Value) * Average: 18.3 External Gain Value 1. Ensure there is not enough water in the load assembly that when the pump is turned to 100% amplitude it sprays. 2. Run the pump with an internal S50_70 waveform up to 100% amplitude. Record the maximum stroke volume. (should be 185 ish) Max Stroke: ml/s 3. Stop the pump. Change the input waveform to External. Select a S50_70 waveform in ViViTest and hit apply. 4. Turn the pump off. Turn it back on and enter Calibration Mode. Enter Calibration menu 2. Turn the amplitude up to 100%. Write down the Max Stroke: Max Stroke: ml/s 5. Adjust the External Gain value until the max stroke in step 4) matches the max stroke in step 2). Record the new External Gain value: External Gain: 18.4 Determining External Pump Calibration Value 1. Turn the amplitude to 0. Exit Calibration Mode. Repeat the following table using the external waveform: Controller Stroke (ml/s) ViViTest Stroke (ml/s) = (ViViTest value / Controller Value) * Average: 2. If different from the internal value. Average these 2 values together. Select the Pump calibration tab and enter the new value. Repeat entering the Acquire Tab of ViViTest process to save it. 3. Run both internal and external S50_70 waveforms at various amplitudes and ensure the stroke volume displayed by the controller matches the ViViTest value within +/- 0.1 or 0.2 ml/s Document No V1.3 ViVitro Labs Inc Page 99 of 118

100 19. Appendix G Calculations Pulse Duplicator System User Manual Crossover points are defined as follows F1 - Flowrate - Beginning of systole Forward Flow F2 - Flowrate - End of systole forward flow F3 - Flowrate Outflow Valve Closing, leakage begins F4 - Flowrate - End of Leakage/Cycle P1 - Pressure Drop - Beginning of Systole Positive Pressure Drop P2 - Pressure Drop - End of Systole Positive Pressure Drop P3 - Pressure Drop - Beginning of Diastole Positive Pressure Drop P4 - Pressure Drop - End of Diastole Positive Pressure Drop (P) Calculated between Positive pressure markers (F) Calculated between Flow markers (H) Calculated between first Pressure and second Flow marker General parameters Cardiac Output (Liters per Minute): Mean Flowrate (F1 to F4) Heart Rate (beats per min): (60 (sec/min)) / Total Period (sec) Total Period = total points between F1 to F4 / sample Rate (samples/sec) Total Period (sec): #samples between F1 to F4/sample Rate (samples/sec) Flow parameters Pump Stroke Volume (ml): (Maximum Position signal Minimum Position signal)* Cross-sectional Area of piston. Calculated Stroke Volume (ml): Cardiac Output (l/min) *1000 (ml/l) / Heart rate (beats/min) Systolic percent of time #samples between maximum and minimum position signal/# points between F1 and F4 Forward flow time #samples between f1 to f2/ sample Rate (samples/sec) [F3 to F4 for Mitral] RMS Forward Flow (ml/sec): (RMS Flowrate (f1 to f2) *1000)/60 (sec/min) [F3 to F4 for Mitral] Mean Forward Flow (ml/sec): Mean Flowrate (f1 to f2) *1000)/60 (sec/min) [F3 to F4 for Mitral] Document No V1.3 ViVitro Labs Inc Page 100 of 118

101 Forward Flow Volume (ml): Flowrate Area (F1 to F2)*1000(ml/Liter)/60(sec/min) [F3 to F4 for Diastolic FF volume] Closing Volume (ml): (flowrate Area (F2 to F3))*1000(ml/Liter)/60(sec/min) [F1 to F2 for Mitral] Leakage Volume (ml): (flowrate Area (F3 to F4))*1000(ml/Liter)/60(sec/min) [F2 to F3 for Mitral] Regurgitant Fraction (RF) (%): (closing volume + leakage Volume)*100/Forward Volume Forward Flow Ratio (%): Forward Flow time *100 / Sample rate (samples/sec) Mean Leakage Flowrate (ml/sec): -1* (Mean Flowrate (F3 to F4)) [F2 to F3 for Mitral] Pressure parameters Mean Pressure Drop [P] (mmhg): (Mean of pressure difference between outflow and inflow of points between P1 to P2) [P3 to P4 for Mitral] Mean Pressure Drop [F] (mmhg): (Mean of pressure difference between outflow and inflow of points between F1 to F2) [F3 to F4 for Mitral] Back Pressure (mmhg): -1* (Mean Pressure Drop (F3 to F4)) Maximum Pressure Drop (mmhg): Maximum Pressure Drop value between Points P1 to P2 Mean Aortic Pressure (mmhg): Mean Aortic/Proximal/Pulmonary Pressure between P1 to P4 without compensation RMS Aortic Pressure (mmhg): RMS of aortic pressure F1 to F4 without compensation Peak Aortic Pressure (mmhg): Peak Aortic Pressure between P1 to P2 [Mitral between P3 to P4] Mean Vent Pressure (mmhg): Mean Vent Pressure between P1 to P4 Peak Vent Pressure (mmhg): Peak Vent Pressure between P1 to P4 Parameters based on pressure and flow E.O.A. (cm^2): RMS Forward Flow (ml/sec)/51.6*(square Root (Mean Pressure Drop/Density)) Document No V1.3 ViVitro Labs Inc Page 101 of 118

102 Parameters based on Energy Energy loss is calculated by integrating the flow times the transvalvular pressure over a relevant flow interval. A conversion factor of is applied to convert the energy from mmhg*ml to millijoules (mj). Ventricular energy (VE) (mj): Ventricular energy is computed using the following integral Where the integral is evaluated using trapezoidal rule and dt= 1/sample rate Forward Energy (FE) (mj): Closing Energy (CE) (mj): Leakage Energy (LE) (mj): Total Energy (mj): Forward Energy+Closing Energy+Leakage Energy Document No V1.3 ViVitro Labs Inc Page 102 of 118

103 20. Appendix H Step By Step Guide 20.1 Definitions Pulse Duplicator System User Manual Symbol Feasibility test Regulatory test BPM SV CO FV Regurgitation % Transvalvular Regurgitant Fraction Total Regurgitant Fraction Definition A test conducted for research and development purposes only. This type of test is not intended for submission to a regulatory body. A test conducted with the intent of submission to a regulatory body (i.e. FDA, CE). The test should fully comply with all applicable regulations. Frequency of operation expressed in terms of beats per minute. Stroke Volume is the amount of fluid ejected from the left ventricle during each beat. Value is calculated from piston area and stroke length. Cardiac Output in liters per minute (LPM) calculated as product of SV and HR. Forward Flow Volume is the volume (ml) of fluid passing through the valve during the forward flow phase of the cycle. Total regurgitation expressed as a percent of the total forward flow [(Closing Volume + Leakage Volume)/FV]. Is defined as the volume of fluid that flows through a valve in the reverse direction during one cycle and is the sum of the closing and leakage volume expressed as a percentage of the stroke volume. This assessment is conducted with sealing applied to the valve to prevent paravalvular leakage. Is defined as the volume of fluid that flows through and around a valve in the reverse direction during one cycle and is the sum of the closing and leakage volume expressed as a percentage of the stroke volume. This assessment includes both transvalvular and paravalvular leakage. EOA Effective orifice area determined by the Gorlin equation (cm 2 ) EOA Where: 51.6 Q rms p Q RMS is the root mean square forward flow (ml/s) during the positive differential pressure period or Aortic or Mitral RMS Flow [P] in ViViTest p is the mean pressure difference measured during the positive differential pressure period (mmhg) or Trans Aortic or Mitral mean pressure [P] in ViViTest ρ is the density of the test fluid (g/cm 3 ) Transaortic pressure Transmitral pressure AO Positive Pressure The P across the aortic valve, the difference in aortic and ventricular pressures. The P across the mitral valve, the difference in atrial and ventricular pressures. The part of a cardiac cycle in which P across the aortic valve is positive [ventricular pressure greater than aortic pressure]. Document No V1.3 ViVitro Labs Inc Page 103 of 118

104 MI Positive Pressure The part of a cardiac cycle in which P across the mitral valve is positive [atrial pressure greater than ventricular pressure]. Test Fluid The fluid that will come into contact with a sample in the Pulse Duplicator. Examples: Saline, Blood Analog, Bovine Blood etc. VIA Companion Valve Viscoelastic Impedance Adapter. The valve located in the opposite position of the valve being tested. For example if an aortic valve is being tested, the valve in the mitral position is the companion valve. By default the companion valve is the ViVitro Spring Loaded Disk Valve. Note: When possible, the companion valve in the mitral test position will be at least one size larger than the valve being tested in the aortic test position. Sample Run Could be valve etc. If the test specimen requires a holder to interface with the testing equipment the entire assembly will be considered a sample. The measurement, recording and subsequent averaging of required parameters for 10 cardiac cycles. Nominal Conditions Test parameters used for acceptance criteria listed in ISO Consisting of 5 LPM at a beat rate of 70/minute and stroke volume of 75 ml/stroke with a 35% systolic duration Summary Test (ref ISO Annex N) The heart valves undergoing testing are mounted in the active test position (aortic/mitral) of the Pulse Duplicator and a companion valve is mounted in the opposite test position. The test loop is filled with a blood analog solution. The valves are then subjected to pulsatile flow operation that is intended to simulate physiologic conditions. Measurements of the various chamber pressures and the flow through the test position are obtained. The results are used to determine the pressure drops through the valve during the forward flow phase of the cycle and the reflux volumes of fluid during the closing and leakage phases of the cycle Cautions Do not turn flow meter on while system is drained. Ensure flow meter is always turned off before draining the system. When flushing pressure ports ensure this is done gradually to ensure pressure limits are not exceeded. Watch ViViTest to get a sense of pressures obtained while flushing. Never operate the Pulse Duplicator without ViViTest in live mode with pressure sensors connected and operating properly. When testing valves always make changes to the system gradually, if anything seems abnormal stop the system and re-evaluate. Document No V1.3 ViVitro Labs Inc Page 104 of 118

105 20.4 Pulsatile Flow Testing 20.5 System Set-Up 20.6 Calibration 20.7 Inserting Valves 20.8 Filling Pulse Duplicator 20.9 Zeroing Tuning Data Capture Moving Markers Recording Data Drain/Remove Valve Data Quality Assurance Reporting Test Results 20.5 System Setup 1. Set-up Pulse Duplicator per User Manual. 2. Mount flow probe in location desired for test, either aortic or mitral. Refer to Pulse Duplicator User Manual for instructions. Note: Ensure that flow probe is oriented such that the side with numbers is the outflow side. 3. Turn on SuperPump controller, ampack, and flow meter to allow electronics to come up to operating temperature 4. If required turn on the Heat bath to allow it to come up to required temperature. Document No V1.3 ViVitro Labs Inc Page 105 of 118

106 20.6 Calibration 1. Start ViViTest software and open the calibration file that the software defaults to. 2. Click tab. 3. Click and follow prompts to take zero voltage reading of all channels. 4. Set up Pulse Duplicator for pressure calibration as per Pulse Duplicator User Manual. 5. Click and calibrate as per prompts. Use a manometer for pressure reference, ensure that calibration is valid. 6. Set up Pulse Duplicator for flow calibration as per Pulse Duplicator User Manual. 7. Remove all air from VIA if installed. 8. Follow Flow Measuring System setup instructions as per Pulse Duplicator User Manual. 9. Click and calibrate as per prompts. 10. If testing on a new system for the first time, or if displayed pump and ViViTest stroke volumes do not align 11. Click and calibrate as per User Manual. 12. Click and save calibration file as YYMMDD_TP-XX-XX.cfg (renaming is optional) 13. Turn off flow probe by turning Probe knob on flow meter to Off 14. To verify that the calibration was done correctly, ensure the flow meter is turned on and the pressure ports are open to the test fluid. The flow signal should read zero and all the pressure tracings should be at the same level as the ventricular pressure as shown below Figure 1. Figure 1 - Calibration verification 15. Drain Pulse Duplicator according to Section Document No V1.3 ViVitro Labs Inc Page 106 of 118

107 20.7 Inserting Valves Do not insert bio-prosthetic valves without immediately filling system with test fluid. Handle valves per manufacturers instructions for use. 1. Insert the valve and holder assembly into the Pulse Duplicator as instructed in the User Manual. Note: Holders can be used with and without a step. If a holder has a step remove the keeper ring as listed in the Pulse Duplicator User Manual. Note: It is recommended that post one is oriented towards the inside the of the Pulse Duplicator i.e. If testing an aortic valve ensure that post 1 is oriented towards the mitral chamber, this will ensure constant high-speed videography. If testing a bi-leaflet valve ensure axis between the leaflets is oriented towards the mitral chamber so gravity will have the same effect on both leaflets. 2. If necessary carefully insert outflow tract of valve into glass aorta 3. Install the companion valve as stated above. 4. For each valve record the valve s manufacturer, serial number, and size in the laboratory notebook as they are tested 20.8 Filling Pulse Duplicator 1. Add test fluid through atrial reservoir. 2. Begin pumping fluid by turning SuperPump up to a stoke volume of ~50ml/stroke. 3. Turn flow probe on by turning Probe knob on the flow meter to + 4. Continue to add test fluid as required. Note: It is recommended that the height of the test fluid in the atrium should fluctuate about the same level during testing. This can be adjusted by either adding/removing air from compliance chambers or adding/removing test fluid. Use reference level marked on atrium as a reference. 5. Remove any air collected in aortic view port or atrium using overflow tube or atrial pressure port respectively. 6. Ensure pressure transducers are free from air bubbles. Flush with a syringe or bleed fluid from the system. 7. Add air to compliance chambers if necessary as this will affect the fluid level in the system 8. Confirm fluid level is appropriate as above 9. Run system until appropriate temperature is reached, measure using calibrated thermometer Zeroing the System 1. This is conducted before each data set is collected to account for any baseline drift on all inputs but most importantly to account for any flow meter drift. 2. Ensure system is at the desired testing temperature and has been running for ~5 minutes 3. The flow meter should be on the + on position 4. Ensure pressure transducers are free from air bubbles. Flush with a syringe or bleed fluid from the system. 5. Stop SuperPump Document No V1.3 ViVitro Labs Inc Page 107 of 118

108 6. Open pressure transducers to atmosphere using stopcocks 7. Allow fluid level to stabilize NOTE: The live voltage readings should have fluctuations less than +/ mv before proceeding. 8. Click twice to take zero reading 9. The flow meter may experience a large drift, but all other values (Aortic, Ventricular, Atrial, dl/dt, and pump) should have a drift < 0.01 mv upon zeroing. If outside this range contact the study director. 10. Open pressure transducers back to fluid 11. Proceed to collecting data promptly to minimize flow meter drift Tuning System 1. Depending on test plan, valves can be tested under a variety of operating conditions. The following outlines the steps to obtain the required testing parameters. 2. With pumped stopped set desired waveform and beat rate in ViViTest, click apply. See Figure 2. Figure 2 Setting Waveform and Beat Rate 3. Increase amplitude on SuperPump controller to desired SV. 4. Visually inspect dl/dt waveform to ensure that appropriate waveform has been selected. 5. Remove any air collected in aortic view port or atrium using overflow tube or atrial pressure port respectively. Ensure overflow tube is closed before proceeding. Air will accumulate in the aortic view port each time the Pulse Duplicator is stopped and will need to be removed every run. 6. Use peripheral resistance knob to adjust either Mean Aortic Pressure or back pressure per the test plan. 7. Adjust root compliance such that test fluid is not rising up silicone tubing. If too much air is added, it will by-pass the aortic root and will need to be flushed according to step Adjust aortic compliance such that test fluid is rising and falling around the approximate half way level of the chamber. If testing at 100 mmhg the aortic pressure should be varying between 120 +/-10 and 80 +/-10 mmhg. 9. Adjust VIA compliance if specified in the test plan. Record any changed in lab notebook. 10. Stop the pump. 11. Re-zero system according to Section Repeat steps through to ensure desired conditions are still being met before proceeding with data capture. Document No V1.3 ViVitro Labs Inc Page 108 of 118

109 20.11 Data Capture This section describes the process for testing a single valve. This will have to be repeated for each valve in the test plan. 1. Install test valve in Pulse Duplicator in position to be tested as per Section Fill system as per Section 20.8 allowing system to come to desired operating conditions 3. Fill in session information in ViViTest: 4. Trial Title: test plan number (TP-xx-xx) 5. Trial Number: 00 (optional) 6. Aortic Valve: valve ID in aortic position 7. Mitral Valve: valve ID in mitral position 8. Operator ID: your name (optional) 9. Ventricle Vol: ml (optional) 10. Fluid Density: 1.01 g/ml if saline is test fluid (or as specified in test plan) 11. Fluid Temp: 37 C (or as specified in test plan) (optional) 12. Flow Probe Site: set to location to test valve 13. Target Valve: set to location of test valve 14. Record the following in lab note book, if not already done: 15. Test fluid used and relevant parameters if specified in the test plan (suggested viscosity and density) 16. Temperature of test fluid 17. VIA compliance volume 18. Companion valve used 19. Ensure test fluid is at desired temperature as specified in the test plan with calibrated thermometer. 20. Tune system to first desired parameter as per Section Once system has stabilized with desired parameters click 22. Once data is collected save in desired test folder using name VALVE ID_RUNXX (optional) 23. Follow Section for aortic or mitral valve. 24. Repeat steps 20 for each test condition in the test plan. 25. If required by the test plan record high speed video of each valve at nominal conditions. 26. Once all conditions are complete proceed to Section to drain the system and remove test valve. 27. Have the study director sign off after the first data collection. 28. Notify QA that a testing audit can be conducted. 29. Repeat above for each valve in test plan. 30. Once all valves have been completed proceed to Section to ensure data quality. 31. Ensure data is being backed-up as appropriate. 32. Once testing is complete have study director sign off. Document No V1.3 ViVitro Labs Inc Page 109 of 118

110 20.12 Moving Markers 1. Once data has been captured the analyze tab will need to be used to extract the relevant flow parameters. The procedure differs whether an aortic or mitral valve is being tested. Aortic Valve Testing 2. Clicking will take you to the screen to analyze collected data. The software will automatically open the most recently collected data file, to open a different file use the navigator on the right side of the screen. 3. Note: When analyzing data in ViViTest, channels can be turned on/off by clicking on the desired channel in the legend below the pressure/flow curve display. This can make it easier to locate crossovers for maker setting. 4. Set AO Positive Pressure by setting ao ve1 to the point where TRANSAORTIC signal first rises above zero and ao ve2 to where it crosses back over zero, see Figure 3. In other words move ao ve1 to the first point at which the ventricular pressure is above the aortic pressure. Set ao ve2 to the last point at which the ventricular pressure is above the aortic pressure. Figure 3 Example of AO Positive Pressure Document No V1.3 ViVitro Labs Inc Page 110 of 118

111 5. Set V1 [aortic forward volume] by moving marker f1 to the point at which Flowmeter signal begins to rise above the zero line and marker f2 to the point at which the Flowmeter signal crosses the zero line again, See Figure 4. Figure 4 Example of flow marker setting 6. The closing volume of a valve will usually be a sharp spike of flow in the negative direction. Create an imaginary line that runs tangent to the Flowmeter signal along the straight portion as it begins to rise back towards the zero line. Set f3 to where this imaginary line would intersect zero if it were to be extended. See Figure 4 and Figure 5 for more detail. Markers F2 to F3 will create V3 or the closing volume. Figure 5 Example of closing volume (from ISO 5840) 7. Set f4 to the point at which the flow begins to rise above zero at the end of the cycle. As this is the end of the cycle it should be similar to where f1 was set but at the beginning of the next cycle on the display. Markers f3 to f4 will create the leakage volume V4 as shown in Figure 4. Document No V1.3 ViVitro Labs Inc Page 111 of 118

112 8. Once this is complete the data can now be recorded, proceed to Section Mitral Valve Testing 9. Clicking on the tab in ViViTest will take you to the screen to analyze collected data. The software will automatically open the most recently collected data file, to open a different file use the navigator on the right side of the screen, 10. Note: When analyzing data in ViViTest channels can be turned on/off by clicking on the desired channel in the legend below the pressure/flow curve display. This can make it easier to locate crossovers for maker setting. 11. Set AO Positive Pressure by setting at ve1 to point where TRANSMITRAL signal first rises above zero and at ve2 to where it crosses back over zero, see Figure 6. In other words move at ve1 to the first point at which the mitral pressure is above the ventricular pressure. Set at ve2 to the last point at which the mitral pressure is above the ventricle pressure. Figure 6 Example of MI Positive Pressure 12. Set V1 [Mitral forward volume] by moving f3 to the point at which Flowmeter signal begins to rise above the zero line and f4 to the point at which the Flowmeter signal crosses the zero line again, See Figure 7. Document No V1.3 ViVitro Labs Inc Page 112 of 118

113 Figure 7 Example of flow marker setting 13. The closing volume of a valve will usually be a sharp spike of flow in the negative direction. Set f1 to the point at which the Flowmeter signal first crosses the zero line on the left side of the screen. Create an imaginary line that follows the tangent of the Flowmeter signal along the straight portion as it begins to rise back towards the zero line. Set f2 to where this imaginary line would intersect zero if it were to be extended. See Figure 5 and Figure 7 for more detail. 14. Once this is complete the data can now be recorded proceed to Section Recoding Data 1. Data should be recorded in the lab notebook in accordance with the lab s procedures. 2. Click the button to bring up the analysis table. 3. Click the button to save.acq file. Use the same file name as used in step Close analysis table. 5. Record the following information in lab notebook for each run of each valve: 6. Run Begin at RUN01 and increment for each test condition 7. If a run at the same condition has to be repeated keep the same number and affix a letter and increment as necessary. (example: RUN03B, RUN03C) 8. Stoke Volume (ml) 9. Cardiac Output (LPM) 10. Mean Aortic Pressure (mmhg) or Back Pressure (mmhg) 11. Closing Volume (ml) 12. Leakage Volume (ml) 13. Regurgitant Fraction (%) 14. EOA (cm 2 ) 15. Waveform (Sine50, Sine30, FDA etc.) Drain / Remove Valve 1. Ensure flow meter is turned off before system is drained. 2. Open ventricle drain and move overflow tube to aortic drain port to speed up draining. 3. Ensure valve is removed promptly upon system draining. 4. Rinse valve if necessary before placing in container. Document No V1.3 ViVitro Labs Inc Page 113 of 118